COMBINATION OF BISPECIFIC FUSION PROTEIN AND ANTI-Her2 ANTIBODY FOR TUMOR TREATMENT

ABSTRACT

The present disclosure provides a use of an immune checkpoint inhibitor in combination with a Her2 inhibitor in the preparation of a medicament for treating tumor in a subject in need thereof, and also provides a pharmaceutical composition comprising an effective amount of said immune checkpoint inhibitor and an effective amount of said Her2 inhibitor, and optionally a pharmaceutically acceptable excipient, as well as a use of the pharmaceutical composition in the preparation of a medicament for treating tumor in a subject in need thereof.

BACKGROUND OF THE INVENTION

The immune system plays an important role in the anti-tumor effect of anti-Her2 antibodies, such as the ADCC effect of anti-Her2 antibodies and is related to T cell function. A patient with a higher level of TILs have a better response to anti-Her2 antibodies. Conversely, patients who don't achieve pathological complete remission (pCR) have increased Treg level, and present to be immunosuppressed.

Animal experiments have also shown that Tratuzumab can cause an increased IFN-γ secretion, thereby induce the PDL1 expression. And the pathway may become one of the mechanisms of Trastuzumab resistance.

Preclinical experiments have confirmed that the therapeutic effect of Her2 target depends on the body's adaptive immune response. Thus, combination with tumor immune-related (target such as PD1/PDL1, CTLA4 or 4-1BB can enhance the anti-tumor effect of drugs synergistically.

SUMMARY OF THE INVENTION

The present disclosure provides a use of an immune checkpoint inhibitor in combination with a Her2 inhibitor in the preparation of a medicament for treating tumor in a subject in need thereof, wherein said immune checkpoint inhibitor is capable of specifically binding to PD-L1 and CTLA4, and also provides a pharmaceutical composition comprising an effective amount of said immune checkpoint inhibitor and an effective amount of said Her2 inhibitor, and optionally a pharmaceutically acceptable excipient, as well as a use of the pharmaceutical composition in the preparation of a medicament for treating tumor in a subject in need thereof.

In one aspect, the present disclosure provides a use of an immune checkpoint inhibitor in combination with a Her2 inhibitor in the preparation of a medicament for treating tumor in a subject in need thereof, wherein said immune checkpoint inhibitor is capable of specifically binding to PD-L1 and CTLA4.

In some embodiments, said immune checkpoint inhibitor is a bispecific antibody or an antigen binding fragment thereof.

In some embodiments, said immune checkpoint inhibitor is a bispecific antibody, and said bispecific antibody is a fully human antibody.

In some embodiments, said immune checkpoint inhibitor is an antigen binding fragment, and said antigen binding fragment comprises Fab, Fab′, F(ab)₂, Fv fragment, F(ab′)₂, scFv, di-scFv and/or dAb.

In some embodiments, said immune checkpoint inhibitor is a dimer, and said dimer formed by two polypeptide chains, with each of said two polypeptide chains comprising an antibody Fc subunit, wherein said dimer comprises two or more immunoglobulin single variable domains (ISVDs), at least one of said ISVDs is specific for PD-L1, and at least one of said ISVDs is specific for CTLA4.

In some embodiments, at least one of said two polypeptide chains comprise both an ISVD specific for PD-L1 and an ISVD specific for CTLA4.

In some embodiments, each of said two polypeptide chains comprises both an ISVD specific for PD-L1 and an ISVD specific for CTLA4.

In some embodiments, for one or both of said two polypeptide chains, said ISVD specific for PD-L1 is fused to said ISVD specific for CTLA4, optionally via a linker.

In some embodiments, for one or both of said two polypeptide chains: said ISVD specific for PD-L1 is fused to said ISVD specific for CTLA4, optionally via a linker; and said ISVD specific for CTLA4 is fused to said antibody Fc subunit, optionally via a linker.

In some embodiments, for one or both of said two polypeptide chains: C terminus of said ISVD specific for PD-L1 is fused to N terminus of said ISVD specific for CTLA4, optionally via a linker; and C terminus of said ISVD specific for CTLA4 is fused to N terminus of said antibody Fc subunit, optionally via a linker.

In some embodiments, for one or both of said two polypeptide chains: said ISVD specific for PD-L1 is fused to said ISVD specific for CTLA4, optionally via a linker; and said ISVD specific for PD-L1 is fused to said antibody Fc subunit, optionally via a linker.

In some embodiments, for one or both of said two polypeptide chains: C terminus of said ISVD specific for CTLA4 is fused to N terminus of said ISVD specific for PD-L1, optionally via a linker; and C terminus of said ISVD specific for PD-L1 is fused to N terminus of said antibody Fc subunit, optionally via a linker.

In some embodiments, said antibody Fc subunit is derived from an IgG Fc subunit.

In some embodiments, said IgG is human IgG1.

In some embodiments, said antibody Fc subunit comprises an amino acid sequence as set forth in any one of SEQ ID NO: 35, 38 and 39.

In some embodiments, said ISVD specific for PD-L1 is capable of binding to N-terminal IgV domain of human PD-L1.

In some embodiments, said ISVD specific for PD-L1 is capable of binding to residues I54, Y56, E58, Q66 and/or R113 of human PD-L1 N-terminal IgV domain, wherein said human PD-L1 N-terminal IgV domain comprises an amino acid sequence as set forth in SEQ ID NO: 64.

In some embodiments, said ISVD specific for PD-L1 is capable of further binding to residues D61, N63, V68, M115, S117, Y123 and/or R125 of human PD-L1 N-terminal IgV domain, wherein said human PD-L1 N-terminal IgV domain comprises an amino acid sequence as set forth in SEQ ID NO: 64.

In some embodiments, said ISVD specific for PD-L1 is capable of binding to a conformational epitope of human PD-L1 N-terminal IgV domain, wherein said conformational epitope comprises residues I54, Y56, E58, Q66 and R113 of said human PD-L1 N-terminal IgV domain, and wherein said human PD-L1 N-terminal IgV domain comprises an amino acid sequence as set forth in SEQ ID NO: 64.

In some embodiments, said ISVD specific for PD-L1 is capable of binding to a conformational epitope of human PD-L1 N-terminal IgV domain, wherein said conformational epitope comprises residues I54, Y56, E58, Q66, R113, D61, N63, V68, M115, S117, Y123 and R125 of said human PD-L1 N-terminal IgV domain, and wherein said human PD-L1 N-terminal IgV domain comprises an amino acid sequence as set forth in SEQ ID NO: 64.

In some embodiments, said ISVD specific for PD-L1 is capable of blocking binding of PD-L1 to PD1.

In some embodiments, said ISVD specific for PD-L1 is capable of blocking binding of PD-L1 to CD80.

In some embodiments, said ISVD specific for PD-L1 cross-competes for binding to PD-L1 with a reference anti-PD-L1 antibody, wherein said reference anti-PD-L1 antibody comprises a heavy chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 1.

In some embodiments, said reference anti-PD-L1 antibody comprises a heavy chain CDR3 comprising an amino acid sequence as set forth in any one of SEQ ID NO: 5 and 9.

In some embodiments, said reference anti-PD-L1 antibody comprises a heavy chain CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 2.

In some embodiments, said reference anti-PD-L1 antibody comprises a heavy chain CDR1 comprising an amino acid sequence as set forth in any one of SEQ ID NO: 3 and 7.

In some embodiments, said reference anti-PD-L1 antibody comprises a heavy chain CDR2 comprising an amino acid sequence as set forth in any one of SEQ ID NO: 4, 8 and 11.

In some embodiments, said reference anti-PD-L1 antibody is an ISVD specific for PD-L1.

In some embodiments, said reference anti-PD-L1 antibody comprises a heavy chain variable domain comprising an amino acid sequence as set forth in any one of SEQ ID NO: 6, 10, 12, 13, 14 and 15.

In some embodiments, said reference anti-PD-L1 antibody comprises a heavy chain variable domain comprising an amino acid sequence as set forth in SEQ ID NO: 6.

In some embodiments, said ISVD specific for PD-L1 comprises a heavy chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 1.

In some embodiments, said ISVD specific for PD-L1 comprises a heavy chain CDR3 comprising an amino acid sequence as set forth in any one of SEQ ID NO: 5 and 9.

In some embodiments, said ISVD specific for PD-L1 comprises a heavy chain CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 2.

T In some embodiments, said ISVD specific for PD-L1 comprises a heavy chain CDR1 comprising an amino acid sequence as set forth in any one of SEQ ID NO: 3 and 7.

In some embodiments, said ISVD specific for PD-L1 comprises a heavy chain CDR2 comprising an amino acid sequence as set forth in any one of SEQ ID NO: 4, 8 and 11.

In some embodiments, said ISVD specific for PD-L1 comprises a heavy chain variable domain comprising an amino acid sequence as set forth in any one of SEQ ID NO: 6, 10, 12, 13, 14 and 15.

In some embodiments, said ISVD specific for PD-L1 comprises a heavy chain variable domain comprising an amino acid sequence as set forth in SEQ ID NO: 6.

In some embodiments, said ISVD specific for CTLA4 is capable of specifically binding to human CTLA4.

In some embodiments, said ISVD specific for CTLA4 is capable of blocking binding of CTLA4 to CD80.

In some embodiments, said ISVD specific for CTLA4 is capable of blocking binding of CTLA4 to CD86.

In some embodiments, said ISVD specific for CTLA4 cross-competes for binding to CTLA4 with a reference anti-CTLA4 antibody, wherein said reference anti-CTLA4 antibody comprises a heavy chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 19.

In some embodiments, reference anti-CTLA4 antibody comprises a heavy chain CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 17.

In some embodiments, said reference anti-CTLA4 antibody comprises a heavy chain CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 16.

In some embodiments, said reference anti-CTLA4 antibody comprises a heavy chain CDR2 comprising an amino acid sequence as set forth in any one of SEQ ID NO: 18, 21 and 23.

In some embodiments, said reference anti-CTLA4 antibody is an ISVD specific for CTLA4.

In some embodiments, said reference anti-CTLA4 antibody comprises a heavy chain variable domain comprising an amino acid sequence as set forth in any one of SEQ ID NO: 20, 22, and 24-32.

In some embodiments, said reference anti-CTLA4 antibody comprises a heavy chain variable domain comprising an amino acid sequence as set forth in SEQ ID NO: 20.

In some embodiments, said ISVD specific for CTLA4 comprises a heavy chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 19.

In some embodiments, said ISVD specific for CTLA4 comprises a heavy chain CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 17.

In some embodiments, said ISVD specific for CTLA4 comprises a heavy chain CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 16.

In some embodiments, said ISVD specific for CTLA4 comprises a heavy chain CDR2 comprising an amino acid sequence as set forth in any one of SEQ ID NO: 18, 21 and 23.

In some embodiments, said ISVD specific for CTLA4 comprises a heavy chain variable domain comprising an amino acid sequence as set forth in any one of SEQ ID NO: 20, 22, and 24-32.

In some embodiments, said ISVD specific for CTLA4 comprises a heavy chain variable domain comprising an amino acid sequence as set forth in SEQ ID NO: 20.

In some embodiments, said dimer is a homodimer.

In some embodiments, said linker comprises an amino acid sequence as set forth in any one of SEQ ID NO: 33-34.

In some embodiments, one or both of said two polypeptide chains comprises an amino acid sequence as set forth in any one of SEQ ID NO: 40-43, 46, 48 and 50.

In some embodiments, one or both of said two polypeptide chains comprises an amino acid sequence as set forth in SEQ ID NO 40.

In some embodiments, said dimer is capable of blocking binding of PD-L1 to PD-1.

In some embodiments, said dimer is capable of blocking binding of PD-L1 to CD80.

In some embodiments, said dimer is capable of blocking binding of CTLA4 to CD80.

In some embodiments, said dimer is capable of blocking binding of CTLA4 to CD86.

In some embodiments, said Her2 inhibitor is capable of inhibiting human Her2.

In some embodiments, said Her2 inhibitor is a Her2 antibody or an antigen binding portion thereof and/or a conjugate thereof.

In some embodiments, a Her2 antibody is selected from a group consisting of Pertuzumab, Trastuzumab and Margetuximab.

In some embodiments, said conjugate is selected from a group consisting of DS8201a and T-DM1.

In some embodiments, said Her2 inhibitor is a bispecific antibody or an antigen binding portion thereof, and is capable of binding to different epitopes of human Her2.

In some embodiments, said Her2 inhibitor is a bispecific antibody or the antigen binding portion thereof, and said bispecific antibody or the antigen binding portion thereof has a common light chain, and wherein said common light chain refers to two light chains having the same sequence.

In some embodiments, heavy chains thereof are capable of correctly assembling with said light chains respectively under physiological conditions or during in vitro protein expression.

In some embodiments, the common light chain is capable of assembling with a heavy chain of Pertuzumab and a heavy chain of Trastuzumab, respectively.

In some embodiments, the common light chain is selected from a light chain of Pertuzumab, a light chain of Trastuzumab, or a mutant thereof.

In some embodiments, a sequence of a variable region of the common light chain comprises a sequence selected from those as set forth in amino acid positions 1 to 107 of SEQ ID NO: 65˜SEQ ID NO: 70.

In some embodiments, heavy chain variable regions thereof are a heavy chain variable region of Pertuzumab and a heavy chain variable region of Trastuzumab, respectively, for example, the heavy chain variable regions comprise sequences as set forth in SEQ ID NO: 87 and SEQ ID NO: 88, respectively.

In some embodiments, Fc fragment sequences of the heavy chains comprise sequences as set forth in SEQ ID: 89 and SEQ ID NO: 90, respectively.

In some embodiments, two heavy chains thereof comprise a sequence as set forth in SEQ ID NO: 83 and SEQ ID NO: 84, respectively.

In some embodiments, said Her2 inhibitor is administrated at dose of 0.01 mg/kg to 100 mg/kg.

In some embodiments, said Her2 inhibitor is administrated at dose of 20 mg/kg to 30 mg/kg.

In some embodiments, said immune checkpoint inhibitor is administrated at dose of 0.01 mg/kg to 100 mg/kg.

In some embodiments, said immune checkpoint inhibitor is administrated at dose of 3 mg/kg to 5 mg/kg.

In some embodiments, said Her2 inhibitor is administrated at a dosing frequency of four times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks or once every twelve weeks.

In some embodiments, said Her2 inhibitor is administrated once every two weeks or once every three weeks or with a loading dose.

In some embodiments, said immune checkpoint inhibitor is administrated at a dosing frequency of four times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks or once every twelve weeks.

In some embodiments, said immune checkpoint inhibitor is administrated once every two weeks or once every three weeks.

In some embodiments, said Her2 inhibitor is administrated intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.

In some embodiments, said Her2 inhibitor is administrated by intravenous administration.

In some embodiments, said immune checkpoint inhibitor is administrated intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.

In some embodiments, said immune checkpoint inhibitor is administrated by intravenous administration.

In some embodiments, said tumor is selected from a group consisting of a solid tumor and a hematologic tumor.

In some embodiments, said tumor is selected from a group consisting of NSCLC, breast cancer, gastric cancer, gastroesophageal junction adenocarcinoma, esophageal adenocarcinoma, colorectal, cervical, ovarian, endometrial, biliary tract, colorectal and urothelial cancer.

In some embodiments, said tumor is selected from a group consisting of Her2 aberrated solid tumor and Her2-positive cancer.

In some embodiments, said subject has been administrated anti-Her2 antibody and/or anti-PD1 agent. In some embodiments, said anti-Her2 antibody comprises trastuzumab.

In some embodiments, said subject has been administrated chemotherapy.

In some embodiments, said chemotherapy comprises first line chemotherapy and/or second line chemotherapy. In some embodiments, said second line chemotherapy comprises paclitaxel plus ramucirumab, paclitaxel, docetaxel and/or irinotecan monotherapy.

In another aspect, the present disclosure provides a pharmaceutical composition comprising an effective amount of said immune checkpoint inhibitor of the present disclosure and an effective amount of said Her2 inhibitor, and optionally a pharmaceutically acceptable excipient.

In another aspect, the present disclosure provides a use of the pharmaceutical composition in the preparation of a medicament for treating tumor in a subject in need thereof.

In some embodiments, said pharmaceutical composition is administrated at a dosing frequency of four times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks or once every twelve weeks.

In some embodiments, said pharmaceutical composition is administrated intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.

In some embodiments, said pharmaceutical composition is administrated at dose of 0.01 mg/kg to 100 mg/kg.

In some embodiments, said tumor selected from a group consisting of a solid tumor and a hematologic tumor.

In some embodiments, said tumor selected from a group consisting of NSCLC, breast cancer, gastric cancer, gastroesophageal junction adenocarcinoma, esophageal adenocarcinoma, colorectal, cervical, ovarian, endometrial, biliary tract, colorectal and urothelial cancer.

In some embodiments, said tumor selected from a group consisting of Her2 aberrated solid tumor and Her2-positive cancer.

In some embodiments, a subject suffering said tumor has been administrated anti-Her2 antibody and/or anti-PD1 agent. In some embodiments, said anti-Her2 antibody comprises trastuzumab.

In some embodiments, said subject has been administrated chemotherapy.

In some embodiments, said chemotherapy comprises first line chemotherapy and/or second line chemotherapy. In some embodiments, said second line chemotherapy comprises paclitaxel plus ramucirumab, paclitaxel, docetaxel and/or irinotecan monotherapy.

Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are employed, and the accompanying drawings (also “figure” and “FIG.” herein), of which:

FIG. 1 illustrates examples of the dimers of the present disclosure.

FIG. 2 illustrate a schematic diagram of heterodimer protein fusion. Panel a illustrates a heterodimer Fc fusion technology, and Panel b illustrates a “Fab” technology.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

The term “antibody,” as used herein, generally refers to an immunoglobulin or a fragment or a derivative thereof, and encompasses any polypeptide comprising an antigen-binding site, regardless whether it is produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, polyspecific, non-specific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and grafted antibodies. Unless otherwise modified by the term “intact,” as in “intact antibodies,” for the purposes of this disclosure, the term “antibody” also includes antibody fragments such as Fab, F(ab′)₂, Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen-binding function, i.e., the ability to bind, for example, CTLA-4, or PD-L1 specifically. Typically, such fragments would comprise an antigen-binding domain.

The term “variable region” or “variable domain” of an antibody, as used herein, generally refers to the amino-terminal domains of the heavy or light chain of an antibody. The variable domains of the heavy chain and light chain may be referred to as “VH” and “VL”, respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen binding sites.

The term “variable”, as used herein, generally refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the entire span of the variable domains. Instead, it is concentrated in three segments called hypervariable regions (CDRs or HVRs) both in the light-chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat et al, Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in the binding of antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

The term “CDR,” “HVR,” or “HV,” as used herein, generally refers to the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops. Generally, antibodies comprise six CDRs; three in the VH (HCDR1, HCDR2, HCDR3), and three in the VL (LCDR1, LCDR2, LCDR3). The ISVD of the present disclosure may only comprise 3 CDRs (e.g., in the VH, HCDR1, HCDR2 and HCDR3). In native antibodies, HCDR3 and LCDR3 display the most diversity of the six CDRs, and HCDR3 in particular is believed to play a unique role in conferring fine specificity to antibodies. See, e.g., Xu et al, Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed, naturally occurring camelid antibodies consisting of a heavy chain only are functional and stable in the absence of light chain. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al, Nature Struct. Biol. 3:733-736 (1996).

A number of CDR delineations are in use and are encompassed herein. The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia refers instead to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The AbM CDRs represent a compromise between the Kabat HVRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software.

The “contact” CDRs are based on an analysis of the available complex crystal structures. The residues from each of these CDRs are noted in Table 1:

TABLE 1 Loop Kabat AbM Chothia Contact LCDR1 L24-L34 L24-L34 L26-L32 L30-L36 LCDR2 L50-L56 L50-L56 L50-L52 L46-L55 LCDR3 L89-L97 L89-L97 L91-L96 L89-L96 HCDR1(Kabat H31-H35B H26-H35B H26-H32 H30-H35B Numbering) HCDR1(Chothia H31-H35 H26-H35 H26-H32 H30-H35 Numbering) HCDR2 H50-H65 H50-H58 H53-H55 H47-H58 HCDR3 H95-H102 H95-H102 H96-H101 H93-H101

CDRs may comprise “extended CDRs” as follows: 24-36 or 24-34 (LCDR1), 46-56 or 50-56 (LCDR2) and 89-97 or 89-96 (LCDR3) in the VL and 26-35 (HCDR1), 50-65 or 49-65 (HCDR2) and 93-102, 94-102, or 95-102 (HCDR3) in the VH. The variable domain residues are numbered according to Kabat et al., supra, for each of these definitions.

The expression “variable-domain residue-numbering as in Kabat” or “amino-acid-position numbering as in Kabat,” and variations thereof, generally refers to the numbering system used for heavy-chain variable domains or light-chain variable domains of the compilation of dimer/polypeptide chain in Kabat et al., supra. The Kabat numbering of residues may be determined for a given polypeptide by alignment at regions of homology of the sequence of the polypeptide with a “standard” Kabat numbered sequence.

“Framework” or “FR” residues are those variable-domain residues other than the CDR residues as herein defined. A “human consensus framework” or “acceptor human framework” is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Examples include for the VL, the subgroup may be subgroup kappa I, kappa II, kappa III or kappa IV as in Kabat et al, supra. Additionally, for the VH, the subgroup may be subgroup I, subgroup II, or subgroup III as in Kabat et al., supra. Alternatively, a human consensus framework can be derived from the above in which particular residues, such as when a human framework residue is selected based on its homology to the donor framework by aligning the donor framework sequence with a collection of various human framework sequences. An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain preexisting amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.

The term “homology,” “homologous” or “sequence identity,” as used herein, generally refers to sequence similarity or interchangeability between two or more polynucleotide sequences or between two or more polypeptide sequences. When using a program (e.g. Emboss Needle or BestFit) to determine sequence identity, similarity or homology between two different amino acid sequences, the default settings may be used, or an appropriate scoring matrix, such as blosum45 or blosum80, may be selected to optimize identity, similarity or homology scores. In some embodiments, polynucleotides that are homologous are those which hybridize under stringent conditions and have at least 60%, at least 65%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, and even 100% sequence identity with a reference sequence. Polypeptides that are homologous may have a sequence identity of at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% with each other when sequences of comparable length are optimally aligned.

The term “percent (%) sequence identity,” as used in the context of polypeptide sequences identified herein, generally refers to the percentage of amino acid residues or nucleotides in a query sequence that are identical with the amino acid residues or nucleotides of a second, reference polypeptide sequence or a portion thereof, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid/nucleotide sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Percent identity may be measured over the length of an entire defined polypeptide/polynucleotide sequence, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide/polynucleotide sequence. It is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.

In the present application, the term “bispecific antibody” refers to an antibody that can respectively bind with two antigens or antigen epitopes, comprising a light chain and a heavy chain of an antibody capable of specifically binding to a first antigen or antigen epitope, and a light chain and a heavy chain of an antibody capable of specifically binding to a second antigen or antigen epitope. In one embodiment, in the bispecific antibody, the antibody light chain capable of specifically binding to a first antigen or antigen epitope and the antibody light chain capable of specifically binding to a second antigen or antigen epitope have the same sequences. In one embodiment, in the bispecific antibody, the antibody heavy chain capable of specifically binding to a first antigen or antigen epitope and the antibody heavy chain capable of specifically binding to a second antigen or antigen epitope have different sequences.

The term “PD-L1,” as used herein, generally refers to the Programmed Death Ligand 1 protein, its functional variant and/or its functional fragments. PD-L1 is also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), and is a protein encoded by the CD274 gene (in human). PD-L1 binds to its receptor, programmed cell death protein 1 (PD-1), which is expressed in activated T cells, B cells, and macrophages (Ishida et al., 1992 EMBO J, 11:3887-3395; Okazaki et al., Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice. Science, 2001; 291: 319-22). The complexation of PD-L1 and PD-1 exerts immunosuppressive effects by inhibiting T cell proliferation and cytokine production of IL-2 and IFN-γ (Freeman et al., Engagement of PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation, J. Exp. Med. 2000, 192:1027-1034; Carter et al., PD-1: PD-L inhibitory pathway affects both CD4(+) and CD8(+) T cells and is overcome by IL-2. Eur. J. Immunol. 2002, 32:634-643). For example, the term “PD-L1” may comprise a polypeptide or a fragment thereof having at least about 85% amino acid sequence identity to NCBI Accession No. Q9NZQ7 and that specifically binds PD1. The term PD-L1 includes the entire PD-L1 ligand, soluble PD-L1 ligand, and fusion proteins comprising a functionally active portion of PD-L1 ligand covalently linked to a second moiety, e.g., a protein domain. Also included within the definition of PD-L1 are variants which vary in amino acid sequence from naturally occurring PD-L1 but which retain the ability to specifically bind to the receptor PD1. Further included within the definition of PD-L1 are variants which enhance the biological activity of PD1. PD-L1 sequences are known in the art and are provided, for example, at GenBank Accession Numbers 29126. The term “PD-L1” as used herein includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1, and analogs having at least one common epitope with hPD-L1. For example, the term “PD-L1” also encompasses PD-L1 from other species, such as other mammals, for example, rat, mouse, rabbit, non-human primate, pig, or bovine. The complete hPD-L1 sequence can be found under GenBank Accession No. 29126.

The term “N-terminal IgV domain of human PD-L1,” as used herein, generally refers to an extracellular domain of human PD-L1 located in its N-terminus. The term “N-terminal IgV domain of human PD-L1” may also refer to epitopes within said domain. The N-terminal IgV domain of the human PD-L1 protein (including the signal peptide) may comprise an amino acid sequence as set forth in SEQ ID NO: 64.

The term “CTLA4,” as used herein, generally refers to the Cytotoxic T-Lymphocyte-Associated protein 4, its functional variant and/or its functional fragments. CTLA4 is an immunoinhibitory receptor belonging to the CD28 family. CTLA4 is expressed exclusively on T cells (CD 4⁺ and CD 8⁺ cells) in vivo, and binds to two ligands, CD80 and CD86 (also called B7-1 and B7-2, respectively). For example, the term “CTLA4” may comprise a polypeptide or a fragment thereof having at least about 85% amino acid sequence identity to NCBI Accession No. AAL07473.1 and that specifically binds to CD80 and/or CD86. The term “CTLA4” includes the entire CTLA4 receptor, its extracellular domain, and fusion proteins comprising a functionally active portion of CTLA4 covalently linked to a second moiety, e.g., a protein domain. Also included within the definition of CTLA4 are variants which vary in amino acid sequence from naturally occurring CTLA4 but which retain the ability to specifically bind to the ligand CD80 and/or CD86. CTLA4 sequences are known in the art and are provided, for example, at GenBank Accession No. 1493. The term “CTLA4” as used herein includes human CTLA4 (hCTLA4), variants, isoforms, and species homologs of hCTLA4, and analogs having at least one common epitope with hCTLA4. For example, the term “CTLA4” also encompasses CTLA4 from other species, such as other mammals, for example, rat, mouse, rabbit, non-human primate, pig, or bovine. The complete hCTLA4 sequence can be found under GenBank Accession No. 1493.

The term “antibody Fc subunit,” as used herein, generally refers to a component of an antibody Fc domain. For example, an antibody Fc domain may be formed by two or more members, and each member may be considered as one Fc subunit. The term “Fc domain,” as used herein, generally refers to an Fc part or Fc fragment of an antibody heavy chain. For example, it may refer to the carboxyl terminal portion of an immunoglobulin heavy chain constant region, or an analog or portion thereof capable of binding an Fc receptor. As is known, each immunoglobulin heavy chain constant region comprises four or five domains. The domains are named sequentially as follows: CH1-hinge-CH2-CH3(-CH4). CH4 is present in IgM, which has no hinge region. The Fc domain or Fc subunit useful in the present disclosure may comprise a CH3 domain. For example, the Fc domain or Fc subunit may comprise a CH2 domain and a CH3 domain. In some embodiments, the Fc domain or Fc subunit may also comprise an immunoglobulin hinge region. For example, the Fc domain or Fc subunit may comprise or consist of, from N-terminus to C-terminus, a CH2 domain and a CH3 domain. In another example, the Fc domain or Fc subunit may comprise or consist of, from N-terminus to C-terminus, an immunoglobulin hinge region, a CH2 domain and a CH3 domain. Amino acid residue positions within the Fc domain or Fc subunit may be determined according to Kabat, E. A. et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication No. 91-3242.

The term “Fc domain”, as used herein, generally refers to a C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy-chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue. Suitable native-sequence Fc regions for use in the antibodies of the invention include human IgG1, IgG2 (IgG2A, IgG2B), IgG3 and IgG4.

Unless indicated otherwise herein, the numbering of the residues in an immunoglobulin chain is that of the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5^(th) Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). The “EU index as in Kabat” refers to the residue numbering of the human IgG1 EU antibody.

The term “dimer,” as used herein, generally refers to a macromolecular complex formed by two, usually non-covalently bound, monomer units. Each monomer unit may be a macromolecule, such as a polypeptide chain or a polynucleotide. The term “homodimer,” as used herein, generally refers to a dimer composed of or formed by two substantially identical monomers, such as two substantially identical polypeptide chains. In some cases, the two monomers of a homodimer may be different at one or more regions or positions, however, such difference does not cause significant alteration in the function or structure of the monomer. For example, one of ordinary skills in the art would consider the difference between the two monomers to be of little or no biological and/or statistical significance within the context of the biological characteristic considered in the present disclosure. The structural/compositional difference between said two monomers may be, for example, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, or less.

The term “fused” or “fusion,” as used herein, generally refers the covalent linkage between two polypeptides. The polypeptides are typically joined via a peptide bond, either directly to each other or via an amino acid linker. Optionally, the peptides can be joined via non-peptide covalent linkages known to those of skill in the art.

The term “fusion protein” as used herein, generally refers to a polypeptide that comprises, or alternatively consists of, an amino acid sequence of a polypeptide fused directly or indirectly (e.g., via a linker) to an amino acid sequence of a heterologous polypeptide (i.e., a polypeptide unrelated to the former polypeptide or the domain thereof).

The term “immunoglobulin single variable domain (ISVD),” as used herein, generally refers to antigen-binding domains or fragments such as VHH domains or VH or VL domains, respectively. The terms antigen-binding molecules or antigen-binding protein are used interchangeably and include also the term Nanobodies. The immunoglobulin single variable domains further are light chain variable domain sequences (e.g., a VL-sequence), or heavy chain variable domain sequences (e.g., a VH-sequence); more specifically, they can be heavy chain variable domain sequences that are derived from a conventional four-chain antibody or heavy chain variable domain sequences that are derived from a heavy chain antibody. Accordingly, the immunoglobulin single variable domains can be domain antibodies, or immunoglobulin sequences that are suitable for use as domain antibodies, single domain antibodies, or immunoglobulin sequences that are suitable for use as single domain antibodies, “dAbs,” or immunoglobulin sequences that are suitable for use as dAbs, or Nanobodies, including but not limited to VHH sequences. The immunoglobulin single variable domain includes fully human, humanized, otherwise sequence optimized or chimeric immunoglobulin sequences. The immunoglobulin single variable domain and structure of an immunoglobulin single variable domain can be considered—without however being limited thereto—to be comprised of four framework regions or “FRs,” which are referred to in the art and herein as “Framework region 1” or “FR1”; as “Framework region 2” or “FR2”; as “Framework region 3” or “FR3”; and as “Framework region 4” or “FR4,” respectively; which framework regions are interrupted by three complementary determining regions or “CDRs,” which are referred to in the art as “Complementarity Determining Region 1” or “CDR1”; as “Complementarity Determining Region 2” or “CDR2”; and as “Complementarity Determining Region 3” or “CDR3,” respectively.

The term “humanized,” as used herein, generally refers to an antibody or a fragment thereof, in which some, most or all of the amino acids outside the CDR domains of a non-human antibody are replaced with corresponding amino acids derived from human immunoglobulins. For example, in a humanized form of an antibody, some, most or all of the amino acids outside the CDR domains have been replaced with amino acids from human immunoglobulins, whereas some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they do not abrogate the ability of the antibody to bind to its specific antigen/epitope. A humanized antibody may retain an antigenic specificity similar to that of the original antibody.

The term “epitope” or “antigenic determinant,” as used herein, generally refers to a site on an antigen to which an antibody bind. Epitopes can be formed both from contiguous amino acids (linear epitope) or noncontiguous amino acids juxtaposed by tertiary folding of a protein (conformational epitopes). Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed (1996).

The term “conformational epitope,” as used herein, generally refers to noncontiguous amino acid residues of the antigen (such as the PD-L1 antigen) that are juxtaposed by tertiary folding of a protein. These noncontiguous amino acid residues may come together on the surface when the polypeptide chain folds to form the native protein. The conformation epitope contains, but is not limited to, the functional epitope.

In the present application, 20 conventional amino acids and abbreviations thereof comply with conventional rules. Reference may be made to, Immunology—A Synthesis (Edition II, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.

The term “functional epitope,” as used herein, generally refers to amino acid residues of an antigen that contribute energetically to the binding of an antibody, i.e. forming an “energetic epitope”. Mutation of any one of the energetically contributing residues of the antigen to alanine will disrupt the binding of the antibody such that the relative K_(D) ratio (K_(D) mutant/K_(D) wildtype) of the antibody may be e.g., greater than 2 folds, such as greater than 3 folds, greater than 4 folds, greater than 6 folds, greater than 10 folds, greater than 20 folds, greater than 30 folds, greater than 40 folds, greater than 50 folds, greater than 60 folds, greater than 70 folds, greater than 80 folds, greater than 90 folds, greater than 100 folds, greater than 150 folds, greater than 200 folds, or more.

The term “extracellular domain,” as used herein, generally refers to part of a protein (e.g., a membrane protein, such as a receptor) protruding from the outer membrane of a cell organelle and/or a cell. If the polypeptide chain crosses the bilayer several times, the extracellular domain comprises loops entwined through the membrane. An extracellular domain may recognize and respond to a specific ligand.

The term “linker,” as used herein, generally refers to a synthetic amino acid sequence that connects or links two polypeptide sequences, e.g., that links two polypeptide domains. A linker may connect two amino acid sequences via peptide bonds. In some embodiments, a linker of the present disclosure connects a biologically active moiety to a second moiety in a linear sequence. For example, a peptide linker may be non-immunogenic and flexible, such as those comprising serine and glycine sequences or repeats of Ala-Ala-Ala. Depending on the particular construct of the dimer, a peptide linker may comprise, e.g., 3-30 (such as at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30) amino acid residues.

The term “N-terminal” may be used interchangeably with “N-terminus,” and as used herein, they generally refer to the amino terminus/end of a polypeptide chain.

The term “C-terminal” may be used interchangeably with “C-terminus,” and as used herein, they generally refer to the carboxyl terminus/end of a polypeptide chain.

The term “about,” as used herein, generally refers to a variation that is within a range of normal tolerance in the art, and generally means within ±10%, such as within 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

The terms “co-administration”, “co-administered” or “co-administering”, as used herein, generally refers that one active ingredient (e.g. a dimer) is administered with another active ingredient (e.g. an immune check point inhibitor). The administration of one active ingredient can be carried out either as one single formulation or as two separate formulations (e.g., one for the dimer and one for the immune check point inhibitor). The co-administration can be simultaneous or sequential in either order.

The term “specifically binds to” or “is specific for”, as used herein, generally refers to measurable and reproducible inter actions such as binding between a target and an antibody, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules. For example, an antibody that specifically binds to a target (which can be an epitope) is an antibody that binds this target with greater affinity, avidity, more readily, and/or with greater duration than it binds to other targets. In one embodiment, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (K_(D)) of <1×10⁻⁶M, <1×10⁻⁷M, <1×10⁻⁸M, <1×10⁻⁹M, or <1×10⁻¹° M. In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved among the protein from different species. In another embodiment, specific binding can include, but does not require exclusive binding.

The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. An IgM antibody consists of 5 of the basic heterotetramer units along with an additional polypeptide called a J chain, and contains 10 antigen binding sites, while IgA antibodies comprise from 2-5 of the basic 4-chain units which can polymerize to form polyvalent assemblages in combination with the J chain. In the case of IgGs, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the α and γ chains and four CH domains for u and c isotypes. Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CH1). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VH and VL together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see e.g., Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6. The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chains designated α, δ, ε, γ and μ, respectively. The γ and α classes are further divided into subclasses on the basis of relatively minor differences in the CH sequence and function, e.g., humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1 and IgK1.

In the present application, the term “antigen binding portion” of the antibody refers to one or more portions of a full-length antibody, the antigen binding portion maintains the ability of binding to an antigen (such as Her2) that is the same as that bound by the antibody, and competes with the full-length antibody for the specific binding to an antigen. General reference is made to Fundamental Immunology, Ch. 7 (Paul, W., ed., edition II, Raven Press, N.Y. (1989), which is incorporated herein by reference in its entirety and for all purposes. The antigen binding portion can be produced using a recombinant DNA technology or through enzymatic or chemical breakage of a full-length antibody. In some cases, the antigen binding portion comprises a polypeptide such as a Fab, a Fab′, a F(ab′)2, a Fd, a Fv, a dAb, a complementary determining region (CDR) fragment, a single-chain antibody (such as a scFv), a chimeric antibody, and a diabody, and it comprises at least the part of the antibody sufficiently endowing the polypeptide with the specific antigen binding ability. The antigen binding portion (such as the above antibody fragment) of the antibody may be obtained from a given antibody (such as monoclonal antibody 2E12) using a conventional technology (such as recombinant DNA technology or enzymatic or chemical breakage process) known to those skilled in the art, and are screened for its specificity in a process that is the same for screening full-length antibodies.

The term “polypeptide chain,” as used herein, generally refers to a macromolecule comprising two or more covalently connected peptides. The peptides within a polypeptide chain may be connected with each other via a peptide bond. Each polypeptide chain may comprise one N-terminus or amino terminus and one C-terminus or carboxy terminus.

The term “CD80,” as used herein, generally refers to a ligand for CD28/CTLA4, also known as B7.1, its functional variant and/or its functional fragments. CD80 is generally expressed on the surface of professional antigen presenting cells (APC). For example, the term “CD80” may comprise a polypeptide or a fragment thereof having at least about 85% amino acid sequence identity to NCBI Accession No. P33681 and that specifically binds CTLA4. Also included within the definition of CD80 are variants which vary in amino acid sequence from naturally occurring CD80 but which retain the ability to specifically bind to CTLA4. Further included within the definition of CD80 are variants which enhance the biological activity of CTLA4. CD80 sequences are known in the art and are provided, for example, at GenBank Accession Numbers P33681. The term “CD80” as used herein includes human CD80 (hCD80), variants, isoforms, and species homologs of hCD80, and analogs having at least one common epitope with hCD80. For example, the term “CD80” also encompasses CD80 from other species, such as other mammals, for example, rat, mouse, rabbit, non-human primate, pig, or bovine. The complete hCD80 sequence can be found under GenBank Accession No. P33681.

The term “CD86,” as used herein, generally refers to a ligand for CD28/CTLA4, also known as B7.2, its functional variant and/or its functional fragments. CD86 is generally expressed on the surface of professional antigen presenting cells (APC). For example, the term “CD86” may comprise a polypeptide or a fragment thereof having at least about 85% amino acid sequence identity to NCBI Accession No. P42081 and that specifically binds CTLA4. Also included within the definition of CD86 are variants which vary in amino acid sequence from naturally occurring CD86 but which retain the ability to specifically bind to CTLA4. Further included within the definition of CD86 are variants which enhance the biological activity of CTLA4. CD86 sequences are known in the art and are provided, for example, at GenBank Accession Numbers U04343. The term “CD86” as used herein includes human CD86 (hCD86), variants, isoforms, and species homologs of hCD86, and analogs having at least one common epitope with hCD86. For example, the term “CD86” also encompasses CD86 from other species, such as other mammals, for example, rat, mouse, rabbit, non-human primate, pig, or bovine. The complete hCD86 sequence can be found under GenBank Accession No. U04343.

The term “PD1,” as used herein, generally refers to programmed death-1 receptor, also known as CD279, its functional variant and/or its functional fragments. PD1 is generally expressed on T cells, B cells, natural killer T cells, activated monocytes and dendritic cells (DCs). PD1 may bind to its ligands PD-L1 and PD-L2. For example, the term “PD1” may comprise a polypeptide or a fragment thereof having at least about 85% amino acid sequence identity to NCBI Accession No P42081 and that specifically binds PD-L1. Also included within the definition of PD1 are variants which vary in amino acid sequence from naturally occurring PD1 but which retain the ability to specifically bind to PD-L1. Further included within the definition of PD1 are variants which enhance the biological activity of PD-L1. PD1 sequences are known in the art and are provided, for example, at GenBank Accession Number Q15116.3. The term “PD1” as used herein includes human PD1 (hPD1), variants, isoforms, and species homologs of hPD1, and analogs having at least one common epitope with hPD1. For example, the term “PD1” also encompasses PD1 from other species, such as other mammals, for example, rat, mouse, rabbit, non-human primate, pig, or bovine. The complete hPD1 sequence can be found under GenBank Accession No. Q15116.3.

The term “blocking”, as used herein, generally refers to an inhibition or reduction of the binding activity between a molecule and its specific binding partner, such as between a ligand and its specific receptor.

The term “blocking antibody” and “antagonist antibody” are used interchangeably herein and generally refers to an antibody that inhibits or reduces a biological activity of the antigen it binds to. In some embodiments, blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen. The PD-L1 specific ISVD or the CTLA4 specific ISVD of the present disclosure may be blocking or antagonistic ISVDs. For example, the PD-L1 specific ISVD of the present disclosure may block the interaction between PD-L1 and its receptor PD-1, and thus the signaling through PD-1 so as to restore a functional response by T-cells from a dysfunctional state to antigen stimulation. The CTLA4 specific ISVD of the present disclosure may block the interaction between CTLA4 and CD80/CD86, and thus the signaling through CTLA4 so as to restore a functional response by T-cells from a dysfunctional state to antigen stimulation.

The term “cross-competes for binding”, “cross-competition”, “cross-block”, “cross-blocked” and “cross-blocking” are used interchangeably herein and generally refers to the ability of an antibody or fragment thereof to interfere with the binding directly or indirectly through allosteric modulation of another antibody of the invention (e.g., the PD-L1 specific ISVD or the CTLA4 specific ISVD of the present disclosure) to the target/antigen (e.g., PD-L1 or CTLA4, respectively). The extent to which an antibody or fragment thereof is able to interfere with the binding of another to the target, and therefore whether it can be said to cross-block or cross-compete according to the invention, can be determined using competition binding assays. One particularly suitable quantitative cross-competition assay uses a FACS-based or an AlphaScreen-based approach to measure competition between the labelled (e.g. His tagged, biotinylated or radioactive labelled) an antibody or fragment thereof and the other an antibody or fragment thereof in terms of their binding to the target. In general, a cross-competing antibody or fragment thereof is for example one which will bind to the target in the cross-competition assay such that, during the assay and in the presence of a second antibody or fragment thereof, the recorded displacement of the immunoglobulin single variable domain or polypeptide according to the invention is up to 100% (e.g. in FACS based competition assay) of the maximum theoretical displacement (e.g. displacement by cold (e.g. unlabeled) antibody or fragment thereof that needs to be cross-blocked) by the to be tested potentially cross-blocking antibody or fragment thereof that is present in a given amount. Preferably, cross-competing antibodies or fragments thereof have a recorded displacement that is between 10% and 100%, such as between 50% to 100%.

The term “substantially reduced,” or “substantially different,” as used herein, generally refers to a sufficiently high degree of difference between two numeric values (generally one associated with a molecule and the other associated with a reference/comparator molecule) such that one of skill in the art would consider the difference between the two values to be of statistical significance within the context of the biological characteristic measured by said values (e.g., K_(D) values). The difference between said two values is, for example, greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and/or greater than about 50% as a function of the value for the reference/comparator molecule.

The term “substantially similar” or “substantially the same,” as used herein, generally refers to a sufficiently high degree of similarity between two numeric values (for example, one associated with a molecule of the present disclosure and the other associated with a reference/comparator molecule), such that one of skill in the art would consider the difference between the two values to be of little or no biological and/or statistical significance within the context of the biological characteristic measured by said values {e.g., K_(D) values). The difference between said two values is, for example, less than about 50%, less than about 40%, less than about 30%, less than about 20%, and/or less than about 10% as a function of the reference/comparator value.

In the present disclosure, an amino acid sequence or nucleotide sequence as set forth in a specific SEQ ID NO. also encompasses homologs or variants thereof having substantially the same function/property thereto. For example, a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity thereto; and/or a variant having one or more (e.g., a few, such as 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2) amino acid or nucleotide addition, deletion or substitution.

The term “tumor,” as used herein, generally refers to tumor growth or metastasis, by any clinically measurable degree. The tumor can be a solid tumor, a hematologic tumor, or a lymphoma. For example, the tumor may be selected from lung cancer (such as non-small-cell lung cancer), breast cancer (such as Triple-Negative Breast Cancer), kidney cancer (such as renal cell carcinoma), melanoma, cervical cancer, uterus cancer, pancreatic cancer, peritoneal carcinoma, ovarian cancer, gastric cancer, gastroesophageal junction adenocarcinoma, esophageal adenocarcinoma, biliary tract, urothelial cancer and colon cancer. The tumor may be advanced or metastatic tumor.

The term “subject,” as used herein, generally refers to a human or non-human animal, including, but not limited to, a cat, dog, horse, pig, cow, sheep, goat, rabbit, mouse, rat, or monkey.

The term “treating” as used herein, generally refers to having a therapeutic effect and at least partially alleviating or abrogating an abnormal condition in the organism. The term “treating” refers to ameliorating a symptom of a medicament condition in a group of patients to whom the medicament is administered relative to a control group that does not receive the medicament. The effect of the treatment can be monitored by measuring a change or an absence of a change in cell phenotype, a change or an absence of a change in cell proliferation, a change or an absence of a change in the tumor size, a change or an absence of a change in tumor size, a change or an absence of a change in a progressive disease, a change or an absence of a change in a stable disease, a change or an absence of a change in a disease control rate, a change or an absence of a change in a partial response. The term “treating” or “treatment” does not necessarily mean total cure. Any alleviation of any undesired symptom of the disease to any extent or the slowing down of the progress of the disease can be considered treatment. Furthermore, treatment may include acts which may worsen the patient's overall feeling of well being or appearance.

The term “pharmaceutically acceptable excipients”, as used herein, includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, and the like and combinations thereof, as would be known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

The term “an effective amount” of a compound of the present disclosure, generally refers to an amount of the compound of the present invention that will elicit the biological or medical response of a subject, for example, reduction or inhibition of an enzyme or a protein activity, or ameliorate symptoms, alleviate conditions, slow or delay disease progression, or prevent a disease, etc.

The terms “antigen binding fragment”, as used herein, generally refers to a part of an antibody molecule that comprises amino acids responsible for the specific binding between antibody and antigen. The part of the antigen that is specifically recognized and bound by the antibody is referred to as the “epitope” as described herein above. As mentioned above, an antigen-binding domain may typically comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH); however, it does not have to comprise both. Fd fragments, for example, have two VH regions and often retain some antigen-binding function of the intact antigen-binding domain. Examples of antigen-binding fragments of an antibody include (1) a Fab fragment, a monovalent fragment having the VL, VH, constant light (CL) and CH1 domains; (2) a F(ab′) 2 fragment, a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge region; (3) an Fd fragment having the two VH and CH1 domains; (4) an Fv fragment having the VL and VH domains of a single arm of an antibody, (5) a dAb fragment (Ward et al., “Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted From Escherichia coli,” Nature 341:544-546 (1989), which is hereby incorporated by reference) which has a VH domain; (6) an isolated complementarity determining region (CDR), and (7) a single chain Fv (scFv), for example, derived from a scFV-library. Although the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv) (see e.g., Huston et al., “Protein Engineering of Antibody Binding Sites: Recovery of Specific Activity in an Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichia coli,” Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988), which is hereby incorporated by reference in its entirety). These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are evaluated for function in the same manner as are intact antibodies.

The term a first agent “in combination with” a second agent, as used herein, generally refers to a co-administration of a first agent and a second agent, which for example may be dissolved or intermixed in the same pharmaceutically acceptable carrier, or administration of a first agent, followed by the second agent, or administration of the second agent, followed by the first agent. The present disclosure, therefore, includes methods of combination therapeutic treatment and combination pharmaceutical compositions.

The term “Her2” as used herein, generally refers to the type I transmembrane protein, also known as c-erbB2, ErbB2 or Neu, belonging to the family of epidermal growth factor receptors. In the context of the present disclosure, the term “Her2” also encompasses homologues, variants and isoforms, including splice isoforms, of Her2. The term “Her2” further encompasses proteins having the sequence of one or more of a Her2 homologue, variant and isoform, as well as fragments of the sequences, provided that the variant proteins (including isoforms), homologous proteins and/or fragments are recognized by one or more Her2 specific antibodies, such as provided as Pertuzumab, Trastuzumab and Margetuximab. The Her2 may be a human Her2. The human Her2 gene is mapped to chromosomal location 17q12, and the genomic sequence of Her2 gene can be found in GenBank at NG 007503.1. In human, there are five Her2 isoforms: A, B, C, D, and E; the term “Her2” is used herein to refer collectively to all Her2 isoforms.

Dimers

In one aspect, the present disclosure provides use of an immune checkpoint inhibitor in combination with a Her2 inhibitor in the preparation of a medicament for treating tumor in a subject in need thereof, wherein said immune checkpoint inhibitor may be capable of specifically binding to PD-L1 and CTLA4.

In some embodiments, the immune checkpoint inhibitor may a dimer. The dimer may be formed by two polypeptide chains, with each of the two polypeptide chains comprising an antibody Fc subunit. For example, the dimer may consist of two polypeptide chains with each polypeptide chain comprising an antibody Fc subunit, and the antibody Fc subunit of one polypeptide chain may associate with the antibody Fc subunit of the other polypeptide chain to form the dimer. In an example, the two polypeptide chains of the dimer do not fuse (e.g., via a peptide linker or by a peptide bond) with each other to become one single polypeptide chain.

The dimer may comprise two or more immunoglobulin single variable domains (ISVDs). For example, one polypeptide chain of the dimer may comprise two or more ISVDs, and the other polypeptide chain of the dimer does not comprise any ISVD. In another example, each of the two polypeptide chains may comprise one or more ISVDs. In yet another example, each of the two polypeptide chains may comprise two or more ISVDs.

At least one of the ISVDs may be specific for PD-L1, and at least one of the ISVDs may be specific for CTLA4. For example, one polypeptide chain of the dimer may comprise one or more ISVDs specific for PD-L1 and one or more ISVDs specific for CTLA4, and the other polypeptide chain of the dimer does not comprise any ISVD. In another example, one polypeptide chain of the dimer may comprise one or more ISVDs specific for PD-L1, and the other polypeptide chain of the dimer may comprise one or more ISVDs specific for CTLA4. In another example, one polypeptide chain of the dimer may comprise one or more ISVDs specific for PD-L1 and one or more ISVDs specific for CTLA4, and the other polypeptide chain of the dimer may comprise one or more ISVDs specific for PD-L1 and/or one or more ISVDs specific for CTLA4.

The one or more ISVDs specific for PD-L1 may be identical or different. The one or more ISVDs specific for CTLA4 may be identical or different.

In some cases, the ISVD specific for PD-L1 does not comprise any antibody light chain CDR. In some cases, the ISVD specific for PD-L1 does not comprise any antibody light chain variable region. In some cases, the ISVD specific for PD-L1 does not comprise any antibody light chain or any fragment thereof. In some cases, the ISVD specific for PD-L1 comprises at least heavy chain CDR3. In some cases, the ISVD specific for PD-L1 comprises heavy chain CDR1. In some cases, the ISVD specific for PD-L1 comprises heavy chain CDR2. In some cases, the ISVD specific for PD-L1 comprises a heavy chain variable region. In some cases, the ISVD specific for PD-L1 is an anti-PD-L1 VHH. The ISVD specific for PD-L1 may be humanized.

In some cases, the ISVD specific for CTLA4 does not comprise any antibody light chain CDR. In some cases, the ISVD specific for CTLA4 does not comprise any antibody light chain variable region. In some cases, the ISVD specific for CTLA4 does not comprise any antibody light chain or any fragment thereof. In some cases, the ISVD specific for CTLA4 comprises at least heavy chain CDR3. In some cases, the ISVD specific for CTLA4 comprises heavy chain CDR1. In some cases, the ISVD specific for CTLA4 comprises heavy chain CDR2. In some cases, the ISVD specific for CTLA4 comprises a heavy chain variable region. In some cases, the ISVD specific for CTLA4 is an anti-CTLA4 VHH. The ISVD specific for CTLA4 may be humanized.

In some cases, at least one of the two polypeptide chains may comprise both an ISVD specific for PD-L1 and an ISVD specific for CTLA4. For example, one of the two polypeptide chains may comprise one or more ISVDs specific for PD-L1 and one or more ISVDs specific for CTLA4. In another example, each of the two polypeptide chains may comprise one or more ISVDs specific for PD-L1 and one or more ISVDs specific for CTLA4.

For one or both of the two polypeptide chains, the ISVD specific for PD-L1 may be fused to the ISVD specific for CTLA4, optionally via a linker. For example, in one or both of the two polypeptide chains, there may be one or more ISVDs specific for PD-L1, and one or more ISVDs specific for CTLA4. When two or more ISVDs specific for PD-L1 are present in a single polypeptide chain, they may be fused to each other (e.g., directly or via a peptide linker), and one or more of them may further be fused to one or more ISVDs specific for CTLA4. When two or more ISVDs specific for CTLA4 are present in a single polypeptide chain, they may be fused to each other (e.g., directly or via a peptide linker), and one or more of them may further be fused to one or more ISVDs specific for PD-L1. One or more linkers (e.g., peptide linker) may be present between any two ISVDs, for example, between two ISVDs specific for PD-L1, between two ISVDs specific for CTLA4, or between one ISVD specific from PD-L1 and one ISVD specific for CTLA4.

For one or both of the two polypeptide chains, the ISVD specific for PD-L1 may be fused to the ISVD specific for CTLA4, optionally via a linker; and the ISVD specific for CTLA4 may in turn be fused to the antibody Fc subunit, optionally via a linker. For example, in a single polypeptide chain, the ISVD specific for PD-L1 may be fused to the ISVD specific for CTLA4 directly (e.g., in frame) or via a linker, and the ISVD specific for CTLA4 may be fused to the antibody Fc subunit directly (e.g., in frame) or via a linker. When there are more than one ISVDs specific for PD-L1 and/or more than one ISVDs specific for CTLA4 in a single polypeptide chain, the ISVDs specific for PD-L1 and the ISVDs specific for CTLA4 may be fused directly or via a linker to each other according to any order, and at least one ISVD specific for CTLA4 may be fused to the antibody Fc subunit directly (e.g., in frame) or via a linker. For example, for one or both of the two polypeptide chains, C terminus of the ISVD specific for PD-L1 may be fused to N terminus of the ISVD specific for CTLA4, optionally via a linker; and C terminus of the ISVD specific for CTLA4 may be fused to N terminus of the antibody Fc subunit, optionally via a linker. For example, in a single polypeptide chain, C terminus of one of the ISVDs specific for PD-L1 may be fused to N terminus of one of the ISVDs specific for CTLA4, either directly (e.g., in frame) or via a linker, and C terminus of one of the ISVDs specific for CTLA4 may be fused to N terminus of the antibody Fc subunit, either directly (e.g., in frame) or via a linker. In an example, when there are more than one ISVDs specific for PD-L1 and/or more than one ISVDs specific for CTLA4 in a single polypeptide chain, the ISVDs specific for PD-L1 and the ISVDs specific for CTLA4 may be fused directly or via a linker to each other according to any order, however, C terminus of at least one ISVD specific for PD-L1 may be fused to N terminus of at least one ISVD specific for CTLA4, either directly (e.g., in frame) or via a linker, and C terminus of at least one ISVD specific for CTLA4 may be fused to N terminus of the antibody Fc subunit, either directly (e.g., in frame) or via a linker.

For one or both of the two polypeptide chains, the ISVD specific for CTLA4 may be fused to the ISVD specific for PD-L1, optionally via a linker; and the ISVD specific for PD-L1 may in turn be fused to the antibody Fc subunit, optionally via a linker. For example, in a single polypeptide chain, the ISVD specific for CTLA4 may be fused to the ISVD specific for PD-L1 directly (e.g., in frame) or via a linker, and the ISVD specific for PD-L1 may be fused to the antibody Fc subunit directly (e.g., in frame) or via a linker. When there are more than one ISVDs specific for PD-L1 and/or more than one ISVDs specific for CTLA4 in a single polypeptide chain, the ISVDs specific for PD-L1 and the ISVDs specific for CTLA4 may be fused directly or via a linker to each other according to any order, and at least one ISVD specific for PD-L1 may be fused to the antibody Fc subunit directly (e.g., in frame) or via a linker. For example, for one or both of the two polypeptide chains, C terminus of the ISVD specific for CTLA4 may be fused to N terminus of the ISVD specific for PD-L1, optionally via a linker; and C terminus of the ISVD specific for PD-L1 may be fused to N terminus of the antibody Fc subunit, optionally via a linker. For example, in a single polypeptide chain, C terminus of one of the ISVDs specific for CTLA4 may be fused to N terminus of one of the ISVDs specific for PD-L1, either directly (e.g., in frame) or via a linker, and C terminus of one of the ISVDs specific for PD-L1 may be fused to N terminus of the antibody Fc subunit, either directly (e.g., in frame) or via a linker. In an example, when there are more than one ISVDs specific for PD-L1 and/or more than one ISVDs specific for CTLA4 in a single polypeptide chain, the ISVDs specific for PD-L1 and the ISVDs specific for CTLA4 may be fused directly or via a linker to each other according to any order, however, C terminus of at least one ISVD specific for CTLA4 may be fused to N terminus of at least one ISVD specific for PD-L1, either directly (e.g., in frame) or via a linker, and C terminus of at least one ISVD specific for PD-L1 may be fused to N terminus of the antibody Fc subunit, either directly (e.g., in frame) or via a linker.

The linker (e.g., a peptide linker) employed in the present application (e.g., as comprised by the dimer of the present application) may be a synthetic amino acid sequence that connects or links two polypeptide sequences, e.g., via peptide bonds. For example, the peptide linker may comprise 1-10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids), 1-15 amino acids (e.g., 1-10, 11, 12, 13, 14 or 15 amino acids), 1-20 amino acids (e.g., 1-15, 16, 17, 18, 19, or 20 amino acids), 1-30 amino acids or more (e.g., 1-20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids). For example, the peptide linker may comprise an amino acid sequence as set forth in any of SEQ ID NO: 33-34.

The antibody Fc subunit may be derived from an IgG Fc subunit. For example, the IgG may be selected from the group consisting of IgG1, IgG2, IgG3 and IgG4. In some embodiments, the IgG is a human IgG1, and the IgG Fc subunit is a human IgG1 Fc subunit. In some embodiments, the Fc subunit comprises an amino acid sequence having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) identity to an amino acid sequence as set forth in any one of SEQ ID NO: 35, 38 and 39. For example, the Fc subunit may comprise an amino acid sequence having one or more (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, or more) amino acid deletion, insertion and/or substitution in the amino acid sequence as set forth in any one of SEQ ID NO: 35, 38 and 39.

In some embodiments, the Fc subunit may be a variant of the IgG Fc subunit (e.g., a variant of the human IgG1 Fc subunit). For example, the variant may comprise one or more amino acid mutations that enhance or reduce the ADCC or CDC activities. As another example, the variant may comprise one or more amino acid mutations that affect FcRn binding activity and/or the half-life of the molecule comprising the variant. As yet another example, the variant may comprise one or more amino acid mutations that affect an interaction (e.g., association) between two or more Fc subunits (or Fc monomers) and/or increase or decrease an efficiency of Fc heterodimer formation, for example, the variant may comprise one or more of the amino acid substitutions as described in CN102558355A, CN103388013A, CN105820251A, or CN106883297A, each of which is incorporated by reference herein.

The ISVD specific for PD-L1 may be capable of specifically binding to human PD-L1. For example, the ISVD specific for PD-L1 may be capable of specifically binding to an epitope in an extracellular domain of the human PD-L1. Such epitopes are known in the art, for example, as shown by Gang Hao et al., J. Mol. Recognit. 2015; 28: 269-276, Zhang et al., Oncotarget. 2017 October; 08 (52): 90215-90224, and Zhang et al., Cell Discov. 2017 Mar. 7; 3:17004.

For example, the ISVD specific for PD-L1 may be capable of binding to N-terminal IgV domain of human PD-L1. The N-terminal IgV domain of human PD-L1 (including the signal peptide) may comprise an amino acid sequence as set forth in SEQ ID NO: 64. In the present disclosure, the ISVD specific for PD-L1 may be capable of binding to residues I54, Y56, E58, Q66 and/or R113 of human PD-L1 N-terminal IgV domain. In a specific embodiment, the ISVD specific for PD-L1 is capable of binding to residues I54, Y56, E58, Q66 and R113 of human PD-L1 N-terminal IgV domain (e.g., amino acid residue I54, Y56, E58, Q66 and/or R113 of SEQ ID NO: 64). The ISVD specific for PD-L1 may be capable of further binding to residues D61, N63, V68, M115, S117, Y123 and/or R125 of human PD-L1 N-terminal IgV domain (e.g., amino acid residue D61, N63, V68, M115, S117, Y123 and/or R125 of SEQ ID NO: 64). In some cases, the ISVD specific for PD-L1 may be capable of binding to residues I54, Y56, E58, Q66, R113, D61, N63, V68, M115, S117, Y123 and/or R125 of human PD-L1 N-terminal IgV domain (e.g., amino acid residue I54, Y56, E58, Q66, R113, D61, N63, V68, M115, S117, Y123 and/or R125 of SEQ ID NO: 64). In some cases, the ISVD specific for PD-L1 is capable of binding to a conformational epitope of human PD-L1 N-terminal IgV domain, the conformational epitope may comprise residues I54, Y56, E58, Q66 and/or R113 of the human PD-L1 N-terminal IgV domain (e.g., amino acid residue I54, Y56, E58, Q66 and/or R113 of SEQ ID NO: 64). In some cases, the ISVD specific for PD-L1 is capable of binding to a conformational epitope of human PD-L1 N-terminal IgV domain, the conformational epitope may comprise residue I54, Y56, E58, Q66, R113, D61, N63, V68, M115, S117, Y123 and/or R125 of the human PD-L1 N-terminal IgV domain (e.g., amino acid residue I54, Y56, E58, Q66, R113, D61, N63, V68, M115, S117, Y123 and/or R125 of SEQ ID NO: 64).

The ISVDs specific for PD-L1 of the present disclosure (e.g., PD-L1 ISVD-9 and the humanized variants thereof) bind to the N-terminal IgV domain of human PD-L1. Taking PD-L1 ISVD-9 as an example, the residue Phe101 of PD-L1 ISVD-9 (SEQ ID NO: 6) interacts with Tyr56 of human PD-L1 N-terminal IgV domain, and when the Tyr56 of human PD-L1 N-terminal IgV domain was substituted by Ala, the binding affinity between PD-L1 ISVD-9 and PD-L1 was reduced by over 200 folds. When the Ile54 of human PD-L1 N-terminal IgV domain was substituted by Ala, the binding affinity between PD-L1 ISVD-9 and PD-L1 was reduced by about 40 folds. The residue Asp99 of PD-L1 ISVD-9 (SEQ ID NO: 6) interacts with Arg113 of human PD-L1 N-terminal IgV domain, and when the Arg113 of human PD-L1 N-terminal IgV domain was substituted by Ala, the binding affinity between PD-L1 ISVD-9 and PD-L1 was reduced by about 90 folds. The residue Ser100 of PD-L1 ISVD-9 (SEQ ID NO: 6) interacts with Glu58 of human PD-L1 N-terminal IgV domain, and when the Glu58 of human PD-L1 N-terminal IgV domain was substituted by Ala, the binding affinity between PD-L1 ISVD-9 and PD-L1 was reduced by about 25 folds. The residue Thr105 of PD-L1 ISVD-9 (SEQ ID NO: 6) interacts with Gln66 of human PD-L1 N-terminal IgV domain, and when the Gln66 of human PD-L1 N-terminal IgV domain was substituted by Ala, the binding affinity between PD-L1 ISVD-9 and PD-L1 was reduced by about 82 folds. In addition, residues D61, N63, V68, M115, S117, Y123 and R125 of human PD-L1 N-terminal IgV domain may be involved in the interaction between PD-L1 ISVD-9 and human PD-L1, substituting these residues with Ala resulted in a reduction of binding affinity by about 2-10 folds. These results are summarized in Table 2 below.

TABLE 2 Effects of Substitutions in human PD-L1 for binding of PD-L1 ISVD-9 Human PD-L1 mutation K_(D) (M) K_(D, mutant)/K_(D, WT) WT 5.92E−09 1 I54A 2.42E−07 40.9 Y56A 1.24E−06 209.5 E58A 1.49E−07 25.2 D61A 1.99E−08 3.4 N63A 2.30E−08 3.9 Q66A 4.88E−07 82.4 V68A 2.76E−08 4.7 R113A 5.34E−07 90.2 M115A 5.51E−08 9.3 S117A 1.26E−08 2.1 Y123A 4.24E−08 7.2 R125A 2.97E−08 5.0

The ISVD specific for PD-L1 may be capable of blocking binding of PD-L1 to PD1. In some cases, the ISVD specific for PD-L1 may be capable of blocking binding of PD-L1 to CD80.

The ISVD specific for PD-L1 may cross-compete for binding to PD-L1 with a reference anti-PD-L1 antibody.

The reference anti-PD-L1 antibody may comprise a heavy chain CDR3. The heavy chain CDR3 may comprise an amino acid sequence as set forth in DSFX₁X₂PTCX₃X₄X₅X₆SSGAFQY (SEQ ID NO: 1), wherein X₁ may be E or G; X2 may be D or Y; X3 may be T or P; X4 may be L or G; X5 may be V or P; and X6 may be T or A. In some cases, the reference anti-PD-L1 antibody may comprise a heavy chain CDR3 comprising an amino acid sequence as set forth in any one of SEQ ID NO: 5 and 9. The reference anti-PD-L1 antibody may also comprise a heavy chain CDR1. The heavy chain CDR1 may comprise an amino acid sequence as set forth in GX₁X₂X₃X₄X₅RCMA (SEQ ID NO: 2), wherein X₁ may be K or N; X2 may be M or I; X3 may be S or I; X4 may be S or R; and X5 may be R or V. For example, the reference anti-PD-L1 antibody may comprise a heavy chain CDR1 comprising an amino acid sequence as set forth in any one of SEQ ID NO: 3 and 7. In some cases, the reference anti-PD-L1 antibody may comprise a heavy chain CDR2. The heavy chain CDR2 may comprise an amino acid sequence as set forth in any one of SEQ ID NO: 4, 8 and 11. In some cases, the reference anti-PD-L1 antibody is an ISVD specific for PD-L1, such as an anti-PD-L1 VHH. The reference anti-PD-L1 antibody may comprise a heavy chain variable domain. The reference anti-PD-L1 antibody may comprise a heavy chain variable domain comprising an amino acid sequence as set forth in any one of SEQ ID NO: 6, 10, 12, 13, 14 and 15. For example, the heavy chain variable domain may comprise an amino acid sequence as set forth in SEQ ID NO: 6.

In the present disclosure, the ISVD specific for PD-L1 (e.g., as comprised in the dimer of the present disclosure) may comprise a heavy chain CDR3. The heavy chain CDR3 may comprise an amino acid sequence as set forth in DSFX₁X₂PTCX₃X₄X₅X₆SSGAFQY (SEQ ID NO: 1), wherein X₁ may be E or G; X2 may be D or Y; X3 may be T or P; X4 may be L or G; X5 may be V or P; and X6 may be T or A. For example, the ISVD specific for PD-L1 may comprise a heavy chain CDR3 comprising an amino acid sequence as set forth in any one of SEQ ID NO: 5 and 9.

For example, the ISVD specific for PD-L1 may comprise a heavy chain CDR3 comprising an amino acid sequence having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) identity to an amino acid sequence as set forth in any one of SEQ ID NO: 5 and 9. In some cases, the heavy chain CDR3 may comprise an amino acid sequence having one or more (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, or more) amino acid deletion, insertion and/or substitution in the sequence as set forth in any one of SEQ ID NOs: 5 and 9.

In the present disclosure, the ISVD specific for PD-L1 (e.g., as comprised in the dimer of the present disclosure) may also comprise a heavy chain CDR1. The heavy chain CDR1 may comprise an amino acid sequence as set forth in GX₁X₂X₃X₄X₅RCMA (SEQ ID NO: 2), wherein X₁ may be K or N; X₂ may be M or I; X₃ may be S or I; X₄ may be S or R; and X₅ may be R or V. For example, the ISVD specific for PD-L1 may comprise a heavy chain CDR1 comprising an amino acid sequence as set forth in any one of SEQ ID NO: 3 and 7.

For example, the ISVD specific for PD-L1 may comprise a heavy chain CDR1 comprising an amino acid sequence having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) identity to an amino acid sequence as set forth in any one of SEQ ID NO: 3 and 7. In some cases, the heavy chain CDR1 may comprise an amino acid sequence having one or more (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, or more) amino acid deletion, insertion and/or substitution in the sequence as set forth in any one of SEQ ID NOs: 3 and 7.

In the present disclosure, the ISVD specific for PD-L1 (e.g., as comprised in the dimer of the present disclosure) may further comprise a heavy chain CDR2. The heavy chain CDR2 may comprise any suitable amino acid sequence. In some case, the ISVD specific for PD-L1 may comprise a heavy chain CDR2 comprising an amino acid sequence as set forth in any one of SEQ ID NO: 4, 8 and 11.

For example, the ISVD specific for PD-L1 may comprise a heavy chain CDR2 comprising an amino acid sequence having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) identity to an amino acid sequence as set forth in any one of SEQ ID NO: 4, 8 and 11. In some cases, the heavy chain CDR2 may comprise an amino acid sequence having one or more (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, or more) amino acid deletion, insertion and/or substitution in the sequence as set forth in any one of SEQ ID NOs: 4, 8 and 11.

In the present disclosure, the ISVD specific for PD-L1 (as comprised in the dimer of the present disclosure) may comprise a heavy chain variable domain. The ISVD specific for PD-L1 may comprise a heavy chain variable domain comprising an amino acid sequence as set forth in any one of SEQ ID NO: 6, 10, 12, 13, 14 and 15. For example, the heavy chain variable domain may comprise an amino acid sequence as set forth in SEQ ID NO: 6.

For example, the ISVD specific for PD-L1 may comprise a heavy chain variable domain comprising an amino acid sequence having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) identity to an amino acid sequence as set forth in any one of SEQ ID NO: 6, 10, 12, 13, 14 and 15. In some cases, the ISVD specific for PD-L1 may comprise a heavy chain variable domain comprising an amino acid sequence having one or more (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, or more) amino acid deletion, insertion and/or substitution in the sequence as set forth in any one of SEQ ID NO: 6, 10, 12, 13, 14 and 15.

In the present disclosure, the ISVD specific for PD-L1 may comprise an amino acid sequence as set forth in any one of SEQ ID NO: 6, 10, 12, 13, 14 and 15. For example, the ISVD specific for PD-L1 (as comprised in the dimer of the present disclosure) may comprise an amino acid sequence as set forth in SEQ ID NO: 6. For example, the ISVD specific for PD-L1 may comprise an amino acid sequence having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) identity to an amino acid sequence as set forth in any one of SEQ ID NO: 6, 10, 12, 13, 14 and 15. In some cases, the ISVD specific for PD-L1 may comprise an amino acid sequence having one or more (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, or more) amino acid deletion, insertion and/or substitution in the sequence as set forth in any one of SEQ ID NO: 6, 10, 12, 13, 14 and 15.

In some cases, the ISVD specific for PD-L1 comprises or consists of a heavy chain variable domain (VH or VHH).

For example, the ISVD specific for PD-L1 may be selected from PD-L1 ISVD-9, PD-L1 ISVD-6, PD-L1 ISVD-m3, PD-L1 ISVD-4, PD-L1 ISVD-11 and PD-L1 ISVD-13.

The ISVD specific for CTLA4 may be capable of specifically binding to human CTLA4. For example, the ISVD specific for CTLA4 may be capable of specifically binding to an epitope in an extracellular domain of the human CTLA4, such an epitope may include those described in CN107400166A, and those described by Udupi A. Ramagopal, et. al., PNAS 2017 May, 114 (21)

The ISVD specific for CTLA4 may be capable of blocking binding of CTLA4 to CD80. In some cases, the ISVD specific for CTLA4 may be capable of blocking binding of CTLA4 to CD86. In some cases, the ISVD specific for CTLA4 may be humanized.

The ISVD specific for CTLA4 may cross-compete for binding to CTLA4 with a reference anti-CTLA4 antibody.

The reference anti-CTLA4 antibody may comprise a heavy chain CDR3. The heavy chain CDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 19. The reference anti-CTLA4 antibody may also comprise a heavy chain CDR1. The heavy chain CDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 17. In some cases, the reference anti-CTLA4 antibody may comprise a heavy chain CDR2. The heavy chain CDR2 may comprise an amino acid sequence as set forth in AIX₁X₂GGGSTYYADSVKG (SEQ ID NO: 16), wherein X₁ may be Y or S; and X₂ may be I or L. For example, the heavy chain CDR2 may comprise an amino acid sequence as set forth in any one of SEQ ID NO: 18, 21 and 23. In some cases, the reference anti-CTLA4 antibody is an ISVD specific for CTLA4, such as an anti-CTLA4 VHH. The reference anti-CTLA4 antibody may comprise a heavy chain variable domain. The reference anti-CTLA4 antibody may comprise a heavy chain variable domain comprising an amino acid sequence as set forth in any one of SEQ ID NO: 20, 22, and 24-32. For example, the heavy chain variable domain may comprise an amino acid sequence as set forth in SEQ ID NO: 20.

In the present disclosure, the ISVD specific for CTLA4 (e.g., as comprised in the dimer of the present disclosure) may comprise a heavy chain CDR3. The heavy chain CDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 19.

In some cases, the ISVD specific for CTLA4 may comprise a heavy chain CDR3 comprising an amino acid sequence having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) identity to an amino acid sequence as set forth in SEQ ID NO: 19. In some cases, the heavy chain CDR3 may comprise an amino acid sequence having one or more (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, or more) amino acid deletion, insertion and/or substitution in the sequence as set forth in SEQ ID NO: 19.

In the present disclosure, the ISVD specific for CTLA4 (e.g., as comprised in the dimer of the present disclosure) may also comprise a heavy chain CDR1. The heavy chain CDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 17.

In some cases, the ISVD specific for CTLA4 may comprise a heavy chain CDR1 comprising an amino acid sequence having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) identity to an amino acid sequence as set forth in SEQ ID NO: 17. In some cases, the heavy chain CDR1 may comprise an amino acid sequence having one or more (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, or more) amino acid deletion, insertion and/or substitution in the sequence as set forth in SEQ ID NOs: 17.

In the present disclosure, the ISVD specific for CTLA4 (e.g., as comprised in the dimer of the present disclosure) may further comprise a heavy chain CDR2. The heavy chain CDR2 may comprise an amino acid sequence as set forth in AIX₁X₂GGGSTYYADSVKG (SEQ ID NO: 16), wherein X₁ may be Y or S; and X2 may be I or L. In some case, the ISVD specific for CTLA4 may comprise a heavy chain CDR2 comprising an amino acid sequence as set forth in any one of SEQ ID NO: 18, 21 and 23.

For example, the ISVD specific for CTLA4 may comprise a heavy chain CDR2 comprising an amino acid sequence having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) identity to an amino acid sequence as set forth in any one of SEQ ID NO: 18, 21 and 23. In some cases, the heavy chain CDR2 may comprise an amino acid sequence having one or more (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, or more) amino acid deletion, insertion and/or substitution in the sequence as set forth in any one of SEQ ID NOs: 18, 21 and 23.

In the present disclosure, the ISVD specific for CTLA4 (as comprised in the dimer of the present disclosure) may comprise a heavy chain variable domain. The ISVD specific for CTLA4 may comprise a heavy chain variable domain comprising an amino acid sequence as set forth in any one of SEQ ID NO: 20, 22, and 24-32. For example, the heavy chain variable domain may comprise an amino acid sequence as set forth in SEQ ID NO: 20.

For example, the ISVD specific for CTLA4 may comprise a heavy chain variable domain comprising an amino acid sequence having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) identity to an amino acid sequence as set forth in any one of SEQ ID NO: 20, 22, and 24-32. In some cases, the ISVD specific for CTLA4 may comprise a heavy chain variable domain comprising an amino acid sequence having one or more (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, or more) amino acid deletion, insertion and/or substitution in the sequence as set forth in any one of SEQ ID NO: 20, 22, and 24-32.

In the present disclosure, the ISVD specific for CTLA4 may comprise an amino acid sequence as set forth in any one of SEQ ID NO: 20, 22, and 24-32. For example, the ISVD specific for CTLA4 (as comprised in the dimer of the present disclosure) may comprise an amino acid sequence as set forth in SEQ ID NO: 20.

For example, the ISVD specific for CTLA4 may comprise an amino acid sequence having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) identity to an amino acid sequence as set forth in any one of SEQ ID NO: 20, 22, and 24-32. In some cases, the ISVD specific for CTLA4 may comprise an amino acid sequence having one or more (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, or more) amino acid deletion, insertion and/or substitution in the sequence as set forth in any one of SEQ ID NO: 20, 22, and 24-32.

In some cases, the ISVD specific for CTLA4 comprises or consists of a heavy chain variable domain (VH or VHH).

For example, the ISVD specific for CTLA4 may be selected from CTLA4 ISVD-34, CTLA4 ISVD-C1, CTLA4 ISVD-13, CTLA4 ISVD-26, CTLA4 ISVD-27, CTLA4 ISVD-28, CTLA4 ISVD-29, CTLA4 ISVD-30, CTLA4 ISVD-31, CTLA4 ISVD-32, and CTLA4 ISVD-33.

For example, the dimer of the present application may comprise or consist of two polypeptide chains. The amino acid sequence of the two polypeptide chains may be identical or different. In some cases, the dimer of the present disclosure may be homodimer.

In the present disclosure, one or both of the two polypeptide chains of the dimer may comprise an amino acid sequence as set forth in any one of claims 40-43, 46, 48 and 50. For example, one or both of the two polypeptide chains of the dimer may comprise an amino acid sequence as set forth in SEQ ID NO: 40.

In specific examples, one or both of the two polypeptide chains of the dimer may comprise an amino acid sequence having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) identity to an amino acid sequence as set forth in any one of SEQ ID NO: 40-43, 46, 48 and 50. In some cases, one or both of the two polypeptide chains of the dimer may comprise an amino acid sequence having one or more (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, or more) amino acid deletion, insertion and/or substitution in the sequence as set forth in any one of SEQ ID NO: 40-43, 46, 48 and 50.

In an example, an ISVD specific for PD-L1 may be fused (directly or indirectly, e.g., via a linker, such as a peptide linker) to an N-terminal amino acid of an ISVD specific for CTLA4 to form a bi-specific binding moiety. Then, one such bi-specific binding moiety may be fused (directly or indirectly, e.g., via a linker, such as a peptide linker) to an N-terminal amino acid of one Fc subunit of the present disclosure to provide one polypeptide chain of the dimer. Then, another such bi-specific binding moiety may be fused (directly or indirectly, e.g., via a linker, such as a peptide linker) to an N-terminal amino acid of another Fc subunit of the present disclosure to provide the other polypeptide chain of the dimer. The two Fc subunits of the two polypeptide chains may associate with each other (e.g., via non-covalent interactions and/or disulfide bond or other covalent bond, in some cases, such covalent bond is not a peptide bond) to form the dimer. The two bi-specific binding moieties may be identical or different. The two Fc subunits may be identical or different.

In some embodiments, the dimer is a proteinaceous homodimer comprising two identical polypeptide chains, with each polypeptide chain comprising one of the bi-specific binding moiety fused to one of the Fc subunits, and the two Fc subunits associate with each other to form the proteinaceous homodimer. The two Fc subunits may associate with each other via non-covalent interactions and/or disulfide bond or other covalent bond, in some cases, such covalent bond is not a peptide bond.

FIGS. 1A-1B provide examples of the dimer of the present disclosure, wherein 1 indicates the ISVD specific for PD-L1, 2 indicates the ISVD specific for CTLA4, 3 indicates the Fc domain comprising the Fc subunits, and 4 indicates the bi-specific binding moiety.

The dimer of the present disclosure may be capable of competing with CD80 and/or CD86 for binding to CTLA4. For example, the competition may be examined in an in vitro experiment using CTLA4 expressing cell lines, such as a CTLA4 expressing HEK293 cell line. As another example, the competition may be examined in an ELISA essay, such as a competition ELISA assay.

The dimer of the present disclosure may be capable of competing with PD1 and/or CD80 for binding to PD-L1. For example, the competition may be examined in an in vitro experiment using PD-L1 expressing cell lines, such as a PD-L1 expressing A375 cell line. As another example, the competition may be examined in an ELISA essay, such as a competition ELISA assay.

The dimer of the present disclosure may be capable of blocking binding of PD-L1 to PD-1. In some cases, the dimer of the present disclosure may be capable of blocking binding of PD-L1 to CD80. In some cases, the dimer of the present disclosure may be capable of blocking binding of CTLA4 to CD80. In some cases, the dimer of the present disclosure may be capable of blocking binding of CTLA4 to CD86.

The dimer of the present disclosure may bind to CTLA4 with a K_(D) of a value no more than about 1×10⁻⁶ M, for example, no more than about 1×10⁻⁷ M, no more than about 1×10⁻⁸ M, no more than about 0.5×10⁻⁸ M, no more than about 1×10⁻⁹ M, no more than about 1×10⁻¹⁰ M or lower.

The dimer of the present disclosure may bind to PD-L1 with a K_(D) of a value no more than about 1×10⁻⁶ M, for example, no more than about 1×10⁻⁷ M, no more than about 1×10⁻⁸ M, no more than about 0.5×10⁻⁸ M, no more than about 1×10⁻⁹ M, no more than about 1×10⁻¹⁰ M or lower.

The dimer of the present disclosure may be capable of stimulating the secretion of an immunoregulator (e.g., IL-2) by immune cells (e.g., PBMC cells).

For example, the dimer of the present disclosure may be selected from aPDL1.9-aCTLA4.34-Fc, aPDL1.9-L-aCTLA4.34-Fc, aCTLA4.34-aPDL1.9-Fc, aCTLA4.34-L-aPDL1.9-Fc, aPDL1.6-aCTLA4.34-Fc, aPDL1.m3-aCTLA4.34-Fc and aPDL1.9-aCTLA4.13-Fc.

For example, the dimer of the present disclosure may comprise the ISVD specific for CTLA4 and the ISVD specific for PDL1. The ISVD specific for PD-L1 may comprise the CDR3 comprising an amino acid sequence as set forth in SEQ ID NO. 5, the CDR2 comprising an amino acid sequence as set forth in SEQ ID NO. 4, the CDR1 comprising an amino acid sequence as set forth in SEQ ID NO. 3. And the ISVD specific for CTLA4 may comprise the CDR3 comprising an amino acid sequence as set forth in SEQ ID NO. 19, the CDR2 comprising an amino acid sequence as set forth in SEQ ID NO. 18, and the CDR1 comprising an amino acid sequence as set forth in SEQ ID NO. 17. And the dimer of the present disclosure may comprise the ISVD specific for PD-L1 comprising an amino acid sequence as set forth in SEQ ID NO.6, and the ISVD specific for CLTA4 comprising an amino acid sequence as set forth in SEQ ID NO. 20. For example, the dimer of the present disclosure may comprise an amino acid sequence of SEQ ID NO.40.

And the dimer of the present disclosure may be named as KN046.

Her2 Inhibitor

In the present disclosure, the Her2 inhibitor may be a Her2 antibody or an antigen binding portion thereof and/or a conjugate thereof.

In some embodiments, the Her2 inhibitor may be a bispecific antibody or the antigen binding portion thereof, wherein the bispecific antibody or the antigen binding portion thereof may have a common light chain, and wherein said common light chain refers to two light chains having the same sequence.

In some embodiments, heavy chains of the bispecific antibody or the antigen binding portion thereof may be capable of correctly assembling with said light chains respectively under physiological conditions or during in vitro protein expression.

The common light chain may be engineered from two original monoclonal antibodies (known monoclonal antibodies) that differed from at least the light chain sequence of one of the two original monoclonal antibodies. In some embodiments, the common light chain may be identical to or modified on the basis of one of the two original monoclonal antibodies (e.g., amino acid sequence modification), and the purpose of the modification is to maintain affinity with the respective antigen or epitope. In some embodiments, the amino acid sequence modification may comprise a mutation, deletion or addition of an amino acid, such as a mutation, deletion or addition of no more than 3 amino acids, preferably no more than 2 amino acids, more preferably no more than 1 amino acid.

In the present application, light chain sequences (especially variable region sequences) of two monoclonal antibodies (namely original antibodies) against different antigens or antigen epitopes may be analyzed and verified to obtain a common light chain capable of assembling with the heavy chains of the two monoclonal antibodies. After assembling with the heavy chains, the common light chain still can specifically bind to the antigens or antigen epitopes directed to by the original monoclonal antibodies.

In the present application, light chain sequences (especially variable region sequences) of two monoclonal antibodies (namely original antibodies) against different antigens or antigen epitopes may be analyzed and verified to obtain a common light chain capable of assembling with the heavy chains of the two monoclonal antibodies. After assembling with the heavy chains, the common light chain still can specifically bind to the antigens or antigen epitopes directed to by the original monoclonal antibodies.

In the present application, the common light chain can be used for expressing the bispecific antibody, and can also be used for expressing a mixture comprising two antibodies; when the bispecific antibody is expressed, the antibody comprises a light chain and a heavy chain which are capable of binding to a first antigen, and a light chain and a heavy chain which may be capable of binding to a second antigen, wherein the sequences of the two light chain are completely the same, namely, is a common light chain.

In the present application, light chain constant regions of the two original antibodies may be of κ type or λ type; the K-type light chain constant region comprises various allotypes, such as Km1, Km2 and Km3; the k-type light chain constant region comprises various allotypes, such as CL1, CL2, CL3, CL6 and CL7.

It is known in the art that the variable region may be crucial for specific binding between the antigen and the antibody, thus, during the process of modifying or obtaining an antibody, the selection and modification of the variable region sequences are critical. Hence, in the present application, in order to obtain the bispecific antibody or the antibody mixture having the common light chain, the variable regions of the common light chain may need to be obtained firstly. After selecting the light chain variable region of one original monoclonal antibody or a mutant thereof as the variable region of the common light chain according to the method discussed above, the constant region of the common light chain is determined. Normally, the common light chain constant region may be determined to be the light chain constant region of the monoclonal antibody from which the common light chain variable region is derived. In some cases, the light chain constant region of the other monoclonal antibody may be determined to be the common light chain constant region. When necessary, the original light chain constant region may be modified (e.g., by addition, deletion or mutation of amino acid, etc.) based on knowledge known in the art to obtain a more suitable constant region of the common light chain, for example, after modification, the common light chain constant region has a better ADCC, CDC, endocytosis, stability, immunogenicity or half-life etc.

In the present application, the heavy chain type of the two original antibodies may be the same or different, preferably, the heavy chains are of the same type. In one embodiment, when preparing the bispecific antibody and the antibody mixture, the sequences of the variable region and the CH1 domain of the heavy chain are unchanged comparing to that of the original antibodies.

In the present application, two arms of the bispecific antibody or the antibodies of the mixture containing two antibodies may be are both derived from two original monoclonal antibodies. When preparing the bispecific antibody or the antibody mixture, only sequences of the light chain variable region would be changed to obtain the common light chain, while sequences of the heavy chain variable region may not need to be changed. In other words, in the bispecific antibody or the antibody mixture prepared, sequences of the antibody heavy chain variable regions may be the same as that of the original antibody, but sequences of at least one light chain variable region shall be different from that of the original antibody.

In the present application, the two original monoclonal antibodies may be selected according to different demands or objectives, for example, the selected two monoclonal antibodies can be against different antigen epitopes of the same antigen; alternatively, one of selected antibodies may bind to a related antigen on the surface of a tumor cell while the other antibody may trigger an immunologic effector cell so as to further kill a cell.

In the present application, when preparing the bispecific antibody, the heavy chain (such as an Fc fragment) may be modified based on technologies known in the art to facilitate formation of the heterodimer protein during antibody expression.

In the present application, when preparing the antibody mixture, the heavy chain (such as an Fc fragment), may be modified based on technologies known in the art so as to facilitate formation of the homodimer protein during antibody expression.

In the present application, technologies for modifying an Fc fragment of the antibody heavy chain to facilitate formation of the homodimer protein or the heterodimer protein are known in the art, for example, reference may be made to, Ridgway, Presta et al. 1996, Carter 2001, Patent CN 102558355A and Patent CN 103388013A.

In the present application, technologies of fusing polypeptides recognizing different antigen epitopes may include but is not limited to, for example, a heterodimer Fc fusing technology as shown in the examples, it can also be an “Fab” technology, see FIG. 2 .

In the present application, the heterodimer Fc fusing technology used in the present application may be based on a “knob”-“hole” model, and it may also be based on a “charge repulsion model”, but is not limited to these two models.

In some embodiments, the heavy chain Fc segment of the bispecific antibody or antigen binding portion thereof may be engineered to be more advantageous for the formation of a heterodimeric protein.

In some embodiments, the two original monoclonal antibodies may be Pertuzumab and Trastuzumab.

In some embodiments, the common light chain may be capable of assembling with a heavy chain of Pertuzumab and a heavy chain of Trastuzumab, respectively.

In some embodiments, the common light chain may be selected from the light chain of Pertuzumab or Trastuzumab or a mutant thereof. In some embodiments, the heavy chain (including the variable region and the constant region) of the bispecific antibody or antigen binding portion thereof may be identical to the two original monoclonal antibodies or may be modified to facilitate formation. Heterodimeric protein; the modification is, for example, engineering the heavy chain Fc segment to facilitate formation of a heterodimeric protein.

In some embodiments, the sequence of the variable region of the common light chain may comprise a sequence selected from amino acids 1 to 107 of SEQ ID NO: 65 to SEQ ID NO: 70. In some embodiments, the sequence of the light chain constant region may comprise the sequence of amino acids 108 to 214 of SEQ ID NO: 65. For example, the variable region of the common light chain of the Her2 inhibitor may be the light chain variable region of Trastuzumab.

In some embodiment, heavy chain variable regions of the antibody or antigen binding portion thereof may be a heavy chain variable region of Pertuzumab and a heavy chain variable region of Trastuzumab, respectively; for example, the heavy chain variable regions may comprise sequences as set forth in SEQ ID NO: 87 and SEQ ID NO: 88, respectively.

In some embodiment, a sequence of a variable region of the two heavy chains may comprise a sequence as set forth in SEQ ID NO: 89 and SEQ ID NO: 90, respectively.

In one embodiment, two heavy chains thereof may comprise a sequence as set forth in SEQ ID NO: 83 and SEQ ID NO: 84, respectively.

For example, the common light chain of the Her2 inhibitor may comprise a sequence of amino acid as set forth in SEQ ID NO: 65; the heavy chain variable regions may comprise sequences as set forth in SEQ ID NO: 87 and SEQ ID NO: 88, respectively, and the variable region of the two heavy chains may comprise a sequence of the amino acid sequence as set forth in SEQ ID NO: 89 and SEQ ID NO: 90, respectively. The two heavy chains thereof may comprise a sequence as set forth in SEQ ID NO: 83 and SEQ ID NO: 84, respectively.

And the Her2 inhibitor of the present disclosure may be named as KN026.

Pharmaceutical Composition and Use

In the present disclosure, provided a pharmaceutical composition comprising an effective amount of said immune checkpoint inhibitor and an effective amount of the Her2 inhibitor of the present disclosure and optionally a pharmaceutically acceptable excipient.

In some embodiments, the term “an effective amount” refers to the amount of the immune checkpoint inhibitor or the Her2 inhibitor of the present disclosure that, when administered to a subject, is effective to at least partially alleviating, inhibiting, preventing and/or ameliorating a condition, or a disorder or a disease. An effective amount of the pharmaceutical composition may be administered for prevention or treatment of disease. The appropriate dosage of the pharmaceutical composition may be determined based on the type of disease to be treated, the type of the pharmaceutical composition, the severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician.

In another aspect, the present disclosure provides a use of the pharmaceutical composition in present disclosure in the preparation of a medicament for treating tumor in a subject.

In another aspect, the present disclosure provides the pharmaceutical composition in present disclosure, for a use in treating tumor in a subject.

In another aspect, the present disclosure provides a method of treating tumor in a subject, comprising the pharmaceutical composition in present disclosure.

In some embodiments, the treatment results in a sustained response in the individual after cessation of the treatment. The medicaments described herein may find use in treating conditions where enhanced immunogenicity is desired such as increasing tumor immunogenicity for the treatment of tumor. The pharmaceutical composition may also enhance immune function in an individual having tumor by administering to the individual an effective amount of the immune checkpoint and the Her2 inhibitor.

In the present disclosure, said tumor may be selected from a group consisting of a solid tumor and a hematologic tumor.

In the present disclosure, said tumor may be selected from a group consisting of NSCLC, breast cancer, gastric cancer, gastroesophageal junction adenocarcinoma, esophageal adenocarcinoma, cervical, ovarian, endometrial, biliary tract, colorectal and urothelial cancer.

In the present disclosure, said tumor may be selected from a group consisting of Her2 aberrated solid tumor and Her2-positive cancer.

In the present disclosure, said subject may have been administrated anti-Her2 antibody and/or anti-PD1 agent.

For example, said anti-Her2 antibody may comprise trastuzumab.

For example, said subject may have been administrated chemotherapy.

In some embodiments, said chemotherapy may comprise a first line chemotherapy and/or a second line chemotherapy. For example, said second line chemotherapy may comprise paclitaxel plus ramucirumab, paclitaxel, docetaxel and/or irinotecan monotherapy.

In present disclosure, said chemotherapy may refer to any treatment against said tumor by chemical agents. Said chemical agent may kill tumor cells, shrink a tumor and/or relieve signs and symptoms of cancer. For example, said chemotherapy may comprise a first line chemotherapy and/or a second line chemotherapy.

In present disclosure, said first line chemotherapy may refer to a chemotherapy regimen or regimens that are generally accepted by the medical establishment for initial treatment of a given type and stage of cancer. For example, said first line chemotherapy may comprise a platinum-based chemotherapy. In some embodiments, said first line platinum-based chemotherapy may comprise chemotherapy with a platinum (P) compound (cisplatin or carboplatin).

In present disclosure, said second line chemotherapy may refer to those tried when the first ones do not work adequately. For example, said second line chemotherapy may comprise paclitaxel plus ramucirumab, paclitaxel, docetaxel and/or irinotecan monotherapy.

In present disclosure, said Her2-positive cancer may refer to a cancer having tumor cells which have expression levels of Her2 higher than normal.

In present disclosure, said Her2 aberrated solid tumor may refer to solid tumor having Her2 aberrations, for example, said solid tumor may have abnormal Her2 gene amplification, Her2 gene mutations, and Her2 protein overexpression.

In present disclosure, said anti-PD1 agent may refer to any agent binds to PD1 and/or blocks the binding between PD1 and PD-L1. For example, said anti-PD1 agent may comprise an anti-PD1 antibody.

In present disclosure, said tumor may comprise said Her2 aberrated solid tumor, with which the subject thereof failed in previous trastuzumab therapy.

In present disclosure, said tumor may comprise Her2-positive GC (gastric cancer) and Her2-positive GEJ (gastroesophageal junction adenocarcinoma) with which the subject thereof failed in at least one prior line of systemic therapy (for example, failed in prior trastuzumab therapy, or failed in both trastuzumab and anti-PD-1 agent therapy).

The pharmaceutical composition may be administered by the same route of administration or by different routes of administration. In some embodiments, the pharmaceutical composition may be administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, said Her2 inhibitor may be administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, said immune checkpoint inhibitor may be administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.

In present disclosure, said Her2 inhibitor may be administrated by intravenous administration.

In present disclosure, said immune checkpoint inhibitor may be administrated by intravenous administration.

As a general proposition, an effective amount of the immune checkpoint inhibitor and an effective amount of the Her2 inhibitor administered to a subject may be in the range of about 0.01 to about 100 mg/kg of subject body weight whether by one or more administrations.

In some embodiments, the Her2 inhibitor may be used is about 0.01 to about 100 mg/kg, about 1 mg/kg to about 80 mg/kg, about 1 mg/kg to about 70 mg/kg, about 1 mg/kg to about 60 mg/kg, about 1 mg/kg to about 50 mg/kg, about 1 mg/kg to about 40 mg/kg, about 1 mg/kg to about 30 mg/kg, about 1 mg/kg to about 100 mg/kg, about 5 mg/kg to about 100 mg/kg, about 10 mg/kg to about 100 mg/kg administered, or about 20 mg/kg to about 100 mg/kg administered, for example. In some embodiments, said Her2 inhibitor may be administered at about 20 to about 30 mg/kg.

In some embodiments, the immune checkpoint inhibitor may be used is about 0.01 to about 100 mg/kg, about 0.01 mg/kg to about 80 mg/kg, about 0.01 mg/kg to about 60 mg/kg, about 0.01 mg/kg to about 40 mg/kg, about 0.01 mg/kg to about 20 mg/kg, about 0.01 mg/kg to about 15 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.01 mg/kg to about 5 mg/kg, about 0.01 mg/kg to about 4 mg/kg, or about 0.01 mg/kg to about 3 mg/kg administered. In some embodiments, said immune checkpoint inhibitor may be administered at about 1 to about 5 mg/kg. For example, said immune checkpoint inhibitor may be administrated at dose of about 3 mg/kg to about 5 mg/kg.

For example, said immune checkpoint inhibitor may be administrated at dose of about 3 mg/kg and said Her2 inhibitor may be administered at about 20 mg/kg or 30 mg/kg. For example, said immune checkpoint inhibitor may be administrated at dose of about 5 mg/kg and said Her2 inhibitor may be administered at about 20 mg/kg.

The dose may be administered as a single dose or as multiple doses (e.g., 2 or 3 doses), such as infusions. The dose of the antibody administered in a combination treatment may be reduced as compared to a single treatment. The progress of this therapy is easily monitored by conventional techniques.

The pharmaceutical composition may be used is about 0.01 to about 100 mg/kg, about 0.01 mg/kg to about 80 mg/kg, about 0.01 mg/kg to about 60 mg/kg, about 0.01 mg/kg to about 40 mg/kg, about 0.01 mg/kg to about 20 mg/kg, about 0.01 mg/kg to about 15 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.01 mg/kg to about 5 mg/kg, about 0.01 mg/kg to about 4 mg/kg, or about 0.01 mg/kg to about 3 mg/kg administered.

The pharmaceutical composition may be administrated to a subject at a dosing frequency of about four times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks, once every twelve weeks, or less frequently so long as a therapeutic response is achieved. In certain embodiments, the Her2 inhibitor may be administrated to a subject at a dosing frequency of about four times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks, once every twelve weeks, or less frequently so long as a therapeutic response is achieved. In certain embodiments, the immune checkpoint may be administrated to a subject at a dosing frequency of about four times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks, once every nine weeks, once every twelve weeks, or less frequently so long as a therapeutic response is achieved.

In present disclosure, said Her2 inhibitor may be administrated once every two weeks or once every three weeks or with a loading dose. In present disclosure, said immune checkpoint inhibitor may be administrated once every two weeks or once every three weeks.

For example, said immune checkpoint inhibitor may be administrated once every two weeks and said Her2 inhibitor may be administrated once every two weeks. For example, said immune checkpoint inhibitor may be administrated once every three weeks and said Her2 inhibitor may be administrated once every two weeks. For example, said immune checkpoint inhibitor may be administrated once every three weeks and said Her2 inhibitor may be administrated once every three weeks.

In the present disclosure, provided a use of the immune checkpoint inhibitor in combination with the Her2 inhibitor in the preparation of a medicament for treating tumor in a subject in need thereof. The use of the term “in combination with” does not restrict the order in which therapies are administered to a subject in need thereof. The immune checkpoint inhibitor can be administered prior to (e.g., 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly or concurrently with, or subsequent to (e.g., 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of the Her2 inhibitor to a subject in need thereof.

EXAMPLES

The following examples are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like.

Example 1 Clinical Study of the Her2 Inhibitor in Combination with the Dimer of the Present Disclosure in Subjects with Her2 Positive Tumor

This is an open-label, phase lb, multicenter clinic study. The purpose of this study is to evaluate the safety and efficacy of the Her2 inhibitor in combination with the dimer of the present disclosure in subjects with Her2 positive tumor.

The study will include a dose escalation phase and a dose expansion phase. The dosage of the Her2 inhibitor in the dose escalation phase will be 20 mg/kg Q2W and 30 mg/kg Q2W; and the dosage of the dimer in the dose escalation phase will be 3 mg/kg Q2W. The mTPI (Modified Toxicity Probability Interval) will be perform in this phase. A mTPI-2 group subjects with increasing or decreasing dosage to determine the maximum tolerated dose (MTD) according to a calculated design. An isotonic regression of mTPI-2 will be applied to the observed DLT (dose limiting toxicity) to estimate the MTD as the dosage most close to the target DLT after the dose escalation phase.

A first dose group (the Her2 inhibitor 6 20 mg/kg Q2W+the dimer 3 mg/kg Q2W) will engage 3-6 subjects. The SMC will be performed after DLT observing and the subjects of the first dose group will be grouped into a second group (the Her2 inhibitor 6 30 mg/kg Q2W+the dimer 3 mg/kg Q2W) if meeting the “ascending to a higher dose” criteria according to the mTPI-2 design table. The second group will engage 3-6 subject and the first dose group will expand to 12 subjects meanwhile. The second dose group will expand to 12 subjects when the subjects meet the “ascending to a higher dose” criteria or “maintain the original dose” criteria and the whole DLT observing of the dose escalation phase will end.

After dose escalation phase, a RP2D (Recommended Phase II Dose) will be determined by the SMC and the study will be in the dose expansion phase. The SMC will determine an original dose, increasing intermediate dose group, or increasing other groups (e.g., fixed-dose administration, different dosing intervals, such as Q3W) at the same dose level according to safety, PK data and/or other data of the Her2 inhibitor and the dimer; or, performing a higher dose study (e.g., the dimer 5 mg/kg).

Primary Study Endpoint:

the dose escalation phase: DLT (dose-limiting toxicity); the dose expansion phase: ORR (objective response rate) determined by RECIST 1.1; and DOR (duration of response). The results show that the Her2 inhibitor in combination with the dimer of the present disclosure will treat the Her2 positive tumor effectively in subjects.

Example 2 Preliminary Safety, Tolerability and Efficacy Results of KN026 (a Her2-Targeted Bispecific Antibody) in Combination with KN046 (an Anti-PD-L1/CTLA-4 Bispecific Antibody) in Patients (Pts) with Her2 Aberrated Solid Tumor

Background:

Her2 potently inhibits innate immunity through cGAS-STING signaling, meanwhile Her2 antibody induced ADCP will also lead to macrophage mediated immune suppression. Both preclinical and clinical studies have suggested a coordination of engagement of innate and adaptive immunity with the combination of an anti-Her2 antibody and an immune checkpoint blockade. KN026 is a novel bispecific antibody that simultaneously binds to two distinct Her2 epitopes. KN046 is a novel bispecific antibody that blocks both PD-L1 interaction with PD-1/CD80 and CTLA-4 interaction with CD80/CD86. Here we reported the interim results from an ongoing phase Ib dose escalation and expansion study assessing the safety, tolerability and preliminary efficacy for KN026 in combination with KN046 in Patients with Her2 aberrated solid tumors.

Methods:

This study enrolled pts with solid tumors who failed available standard of care, Her2 aberration status confirmed locally (Her2 mutation, Her2 amplification and/or Her2 overexpression). Eligible pts received combination of KN026 and KN046 at three dose levels until disease progression, unacceptable toxicity or withdrawal of informed consent (DL1: KN026 20 mg/kg Q2W+KN046 3 mg/kg Q2W; DL2: KN026 20 mg/kg Q2W with loading on Days 1, 8 of Cycle 1+KN046 5 mg/kg Q3W; DL3: KN026 30 mg/kg Q3W with loading on Days 1, 8 of Cycle 1+KN046 5 mg/kg Q3W). Tumor response was evaluated Q8W per RECIST 1.1. Primary endpoint was DLT and key secondary endpoints were efficacy parameters (ORR, DOR, PFS).

Results:

As of the Sep. 8, 2020, 25 pts were enrolled into DL1 (n=20, 3 for dose escalation), DL2 (n=3) and DL3 (n=2) (mGC/GEJ 15 pts; mCRC 8 pts; other solid tumors 2 pts). 15 pts remained on the study treatment and 10 pts discontinued treatment due to disease progression (n=5), death (n=2) and other reasons (n=3). 18 pts had Her2-positive status (12 of 18 failed previous trastuzumab therapy), 2 pts had Her2 mutation and 5 pts had Her2 low expression (without FISH amplification). No DLTs were observed. No pts experienced LVEF decreased or other clinically meaningful cardiac AEs. Treatment-related TEAEs occurred in 23 (92%) pts, of which 6 (24%) pts experienced grade 3 or above treatment-related TEAEs. 11 (44%) pts experienced irAEs, majority were of grade 1 or 2 except that 1 patient experienced grade 3 immune-mediated endocrinopathy. The most common (frequency≥15%) KN026 or KN046 related TEAEs were infusion related reaction (n=11, 44.0%), anaemia (n=9, 36.0%), white blood cell count decreased (n=6, 24.0%), diarrhea (n=5, 20.0%), AST increased (n=5, 20.0%), platelet count decreased (n=5, 20.0%), rash (n=5, 20.0%) and ALT increased (n=4, 16.0%). The objective response rate in pts with Her2-positive tumors (n=14 efficacy evaluable pts) was 9/14 (64.3%, 95% CI 35.187.2%) and disease control rate 13/14 (92.9%, 95% CI 66.199.8%). 4 out of 5 pts with Her2 mutation or low expression achieved SD including one patient with SD for more than 24 weeks. 2 death cases due to disease progression were reported, both only received one cycle of KN026 plus KN046 due to COVID-19 restriction.

Conclusions: KN026 combined with KN046 is well tolerated and has demonstrated profound anti-tumor activity in Her2-positive solid tumors preliminarily.

Clinical Trial Information: NCT04040699

Example 3 Preliminary Breakthrough Therapy Designation Request (BTDR) Advice

This document will be used as a basis for the Division to comment on whether a request for a Breakthrough Therapy Designation (BTD) is appropriate, at this time, may be too preliminary, or does not currently meet the BTD criteria.

1. Provide Information Related to Whether the Indication is Serious and Life-Threatening. Briefly Describe the Indication and the Disease for which the Product is Intended:

Indication which the product is intended is Her2-positive (Her2 IHC3+ or Her2 IHC2+/FISH+) gastric/gastroesophageal junction adenocarcinoma and esophageal adenocarcinoma (GC/GEJ/EAC).

2. Briefly Describe the Drug, the Drug's Mechanism of Action (if Known), the Drug's Relation to Existing Therapy(ies):

KN026 is a Her2 bispecific antibody simultaneously targeting Her2 domains II and IV; KN046 is a PD-L1/CTLA-4 bispecific antibody blocking PD1/PD-L1 and CTLA-4 pathways.

3. Briefly Describe Available Therapies, if any:

Fluoropyrimidine, platinum and trastuzumab based regimen is commonly used as the first line therapy. Available second or later line therapies include paclitaxel plus ramucirumab, paclitaxel, docetaxel or irinotecan monotherapy, and best supportive care, inducing approx. 15-25% objective response rate. Median overall survival is about 8-9 months at second line and 4-6 month at late line with large unmet medical need.

4. Provide Information Related to the Preliminary Clinical Evidence*, Including Trial Design, Trial Endpoints, Treatment Groups, and Number of Subjects Enrolled:

KN046-IST-02 (NCT04040699) is an ongoing dose escalation and expansion study to evaluate efficacy and safety of KN046 plus KN026 in Her2-positive solid tumors. As of 3 Sep. 2020, 14 patients with Her2-positive solid tumors were enrolled and evaluable for efficacy, including 9 patients with Her2-positive GC/GEJ. 64.3% (9/14) response rate was observed in Her2-positive solid tumors and 66.7% (6/9) response rate in Her2-positive GC/GEJ. All 14 patients have failed at least one prior line of systemic therapy; 10 patients have failed prior trastuzumab therapy where 60% (6/10) objective response rate was observed; 2 patients failed both trastuzumab and anti-PD-1 agent with one partial response and one stable disease.

For example, for Oncology/Hematology products, preliminary clinical evidence could include response rates, duration of response, and extent of prior therapies.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby. 

What is claimed is:
 1. A method for treating tumor(s) in a subject in need thereof, comprising administering to said subject an immune checkpoint inhibitor in combination with a Her2 inhibitor, wherein said immune checkpoint inhibitor is capable of specifically binding to PD-L1 (programmed death-ligand 1) and CTLA4 (cytotoxic T-lymphocyte associated protein 4).
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 5. The method according to claim 1, wherein said immune checkpoint inhibitor is a dimer, and said dimer formed by two polypeptide chains, with each of said two polypeptide chains comprising an antibody Fc subunit, wherein said dimer comprises two or more immunoglobulin single variable domains (ISVDs), at least one of said ISVDs is specific for PD-L1, and at least one of said ISVDs is specific for CTLA4.
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 8. The method according to claim 5, wherein for one or both of said two polypeptide chains, said ISVD specific for PD-L1 is fused to said ISVD specific for CTLA4, optionally via a linker.
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 16. The method according to claim 5, wherein said ISVD specific for PD-L1 is capable of binding to N-terminal IgV (immunoglobulin variable) domain of human PD-L1.
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 20. The method according to claim 5, wherein said ISVD specific for PD-L1 is capable of binding to a conformational epitope of human PD-L1 N-terminal IgV domain, wherein said conformational epitope comprises residues I54, Y56, E58, Q66, R113, D61, N63, V68, M115, S117, Y123 and R125 of said human PD-L1 N-terminal IgV domain, and wherein said human PD-L1 N-terminal IgV domain comprises an amino acid sequence as set forth in SEQ ID NO:
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 31. The method according to claim 5, wherein said ISVD specific for PD-L1 comprises a heavy chain CDR3 (complementarity determining region) comprising an amino acid sequence as set forth in SEQ ID NO: 1, said ISVD specific for PD-L1 comprises a heavy chain CDR2 comprising an amino acid sequence as set forth in any one of SEQ ID NO: 4, 8 and 11, and said ISVD specific for PD-L1 comprises a heavy chain CDR1 comprising an amino acid sequence as set forth in SEQ ID NO:
 2. 32. The method according to claim 5, wherein said ISVD specific for PD-L1 comprises a heavy chain CDR3 comprising an amino acid sequence as set forth in any one of SEQ ID NO: 5 and 9, said ISVD specific for PD-L1 comprises a heavy chain CDR2 comprising an amino acid sequence as set forth in any one of SEQ ID NO: 4, 8 and 11, and said ISVD specific for PD-L1 comprises a heavy chain CDR1 comprising an amino acid sequence as set forth in any one of SEQ ID NO: 3 and
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 36. The method according to claim 5, wherein said ISVD specific for PD-L1 comprises a heavy chain variable domain comprising an amino acid sequence as set forth in any one of SEQ ID NO: 6, 10, 12, 13, 14 and
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 48. The method according to claim 5, wherein said ISVD specific for CTLA4 comprises a heavy chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 19, said ISVD specific for CTLA4 comprises a heavy chain CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 16, and said ISVD specific for CTLA4 comprises a heavy chain CDR1 comprising an amino acid sequence as set forth in SEQ ID NO:
 17. 49. The method according to claim 5, wherein said ISVD specific for CTLA4 comprises a heavy chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 19, said ISVD specific for CTLA4 comprises a heavy chain CDR2 comprising an amino acid sequence as set forth in any one of SEQ ID NO: 18, 21 and 23, and said ISVD specific for CTLA4 comprises a heavy chain CDR1 comprising an amino acid sequence as set forth in SEQ ID NO:
 17. 50. (canceled)
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 52. The method according to claim 5, wherein said ISVD specific for CTLA4 comprises a heavy chain variable domain comprising an amino acid sequence as set forth in any one of SEQ ID NO: 20, 22, and 24-32.
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 56. The method according to claim 5, wherein one or both of said two polypeptide chains comprises an amino acid sequence as set forth in any one of SEQ ID NO: 40-43, 46, 48 and
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 71. The method according to claim 1, wherein a sequence of a variable region of the common light chain of said Her2 inhibitor comprises a sequence selected from those as set forth in amino acid positions 1 to 107 of SEQ ID NO: 65 to SEQ ID NO:
 70. 72. The method according to claim 1, wherein heavy chain variable regions of said Her2 inhibitor thereof are a heavy chain variable region of Pertuzumab and a heavy chain variable region of Trastuzumab, respectively, for example, the heavy chain variable regions comprise sequences as set forth in SEQ ID NO: 87 and SEQ ID NO: 88, respectively.
 73. (canceled)
 74. The method according to claim 1, wherein two heavy chains of said Her2 inhibitor thereof comprise a sequence as set forth in SEQ ID NO: 83 and SEQ ID NO: 84, respectively.
 75. The method according to claim 1, wherein said Her2 inhibitor is administrated at the dose of 0.01 mg/kg to 100 mg/kg, and/or said immune checkpoint inhibitor is administrated at dose of 0.01 mg/kg to 100 mg/kg.
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 79. The method according to claim 1, wherein said Her2 inhibitor is administrated at a dosing frequency of four times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks or once every twelve weeks, and/or said immune checkpoint inhibitor is administrated at a dosing frequency of about four times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks, once every nine weeks, or once every twelve weeks.
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 87. The method according to claim 1, wherein said tumor is selected from a group consisting of a solid tumor and a hematologic tumor.
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 95. A pharmaceutical composition comprising an effective amount of said immune checkpoint inhibitor and an effective amount of said Her2 inhibitor according to claim 1, and optionally a pharmaceutically acceptable excipient.
 96. A method for treating tumor in a subject in need thereof, comprising administering to said subject the pharmaceutical composition according to claim
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